Systemin

ABSTRACT

Systemin, an 18 amino acid peptide hormone and first polypeptide hormone found in plants, induces expression of defense genes in plants wounded mechanically or by predators including herbivores, insects, bacteria and viruses. The precursor for systemin is encoded as a 200 amino acid prosystemin molecule that has the systemin peptide sequence located near the carboxyl-terminus. Both a 951 bp cDNA for prosystemin and 4526 bp genomic DNA were cloned and the organization of the gene was determined. Transgenic plants constructed with antisense prosystemin DNA fail to mount a defensive response to wounding. Transgenic plants constructed with increased copy number of prosystemin genes exhibit increased resistance to wounding. Insect larval that feed on transgenic plants constructed with increased copy number of prosystemin genes exhibit decreased growth weight compared to larval that feed on wild type plants. A tomato systemin polypeptide has an amino acid sequence NH 3  -AVQSKPPSKRDPPKMQTD-COO-.

GOVERNMENT SPONSORSHIP

This invention was made with government support under grant numberDCB9104542 awarded by the National Science Foundation. The governmenthas certain rights in the invention.

RELATED APPLICATIONS

The present application claims priority under 35 U.S.C. §120 from PCTInternational Application No. PCT/US93/02428, filed Mar. 18, 1993, whichis a continuation-in-part of U.S. Ser. No. 07/855,412, filed Mar. 19,1992, now U.S. Pat. No. 5,378,819, which is a continuation-in-part ofU.S. Ser. No. 07/528,956, filed May 25, 1990, now abandoned, and acontinuation-in-part of PCT International Application No.PCT/US91/03685.

FIELD OF THE INVENTION

This invention relates to methods and materials for inducing plantdefense mechanisms. More particularly, this invention relates to methodsfor inducing the production of plant defense proteins, such asproteinase inhibitors, and to methods of regulating resistance topredators, herbivores, insect, pathogen or virus in plants by inducingor suppressing the expression of genes encoding systemin or prosystemin.

BACKGROUND OF THE INVENTION

Damage to crops by predators (i.e., insects, herbivores, and pathogens,including fungi, bacteria, and viruses), results in substantial annuallosses in agricultural production. Man has created and employed a widerange of chemicals in attempting to reduce damage to plant crops. Manyenvironmental problems have been created by the widespread use ofchemicals that may only provide a transient level of protection forcrops. Chemicals also suffer from the disadvantage that all organisms inan area may be indiscriminately treated, causing needless damage to manybeneficial organisms. Many chemicals are also potentially toxic to manand animals.

Attempts to reduce crop damage have included selective breeding forresistance, but resistance traits can frequently be controlled by manygenes making it difficult (or impossible) to genetically select adesired attribute. Decreased crop yields are also commonly encounteredin resistance strains. Accordingly, there exists a strong need forcompositions and processes to improve the resistance of plants underattack by herbivores.

Plants have evolved inducible defensive mechanisms that respond toattacks by predators (C. A. Ryan, 1990, Ann. Rev. Phytopathol. 28:425;D. J. Bowles, 1990, Ann. Rev. Biochem, 59:873; M. Chessin and A. E.Zipf, 1990, The Botanical Review 56:193; D. L. Dreyer and B. C.Campbell, 1987, Plant, Cell and Environ. 10:353). One mechanism involvessystemic synthesis of serine proteinase inhibitors that are accumulatedat distal tissue sites in plants. The proteinases can inhibit thedigestive enzymes of insects and microorganisms (T. R. Green and C. A.Ryan, 1972, Science 175:776; C. A. Ryan, 1978, TIBS 3(7):148; V. A.Hilder, A. M. R. Gatehouse, S. E. Sheerman, R. F. Barker, D. Boulter,1987 Nature 330:160; R. Johnson, J. Narvaez, G. An, C. A. Ryan, 1989,Proc. Natl. Acad. Sci. U.S.A. 86:9871). Proteinase inhibitors can bedetrimental to the growth and development of insects from a variety ofgenera including Heliothis, Spodoptera, Diabiotica and Tribolium (Ryan,supra; Broadway, supra; Rechsteiner, supra). Several families ofpolypeptides have been described that inhibit serine proteinases,including: the Kunitz family (e.g., Soybean trypsin inhibitor); theBowman-Birk family; (e.g., Soybean proteinase inhibitor); the Potato Iand Potato II families; the Barley trypsin inhibitor family; and, theSquash inhibitor family.

Wounding of plants by animals, including insects, and pathogens ormechanical damage reportedly induces transcriptional activation ofproteinase inhibitor genes and protein synthesis (J. S. Graham, G. Hall,G. Pearce, C. A. Ryan, 1986, Planta 169:399). The latter wound-responsehas reportedly been described in a variety of species including; tomato(J. S. Graham, G. Pearce, J. Merryweather, K. Titani, L. Ericsson, C. A.Ryan, 1985, J. Biol. Chem. 260(11):6555; J. S. Graham, G. Pearce, J.Merryweather, K. Titani, L. H. Ericsson, C. A. Ryan, 1985, J. Biol.Chem. 260(11):6561), potato (C. A. Ryan, 1968, Plant Physiol. 43:1880),alfalfa (W. E. Brown and C. A. Ryan, 1984, Biochemistry 23:3418; W. E.Brown, K. Takio, K. Titani, C. A. Ryan, 1985, Biochemistry 24:2105),cucurbits (D. Roby, A. Toppan, M. T. Esquerre-Tugaye, 1987, Physiol.Mol. Pl. Pathol. 30:6453) and poplar trees (H. D. Bradshaw, J. B.Hoflick, T. J. Parsons, H. R. G. Clarke, 1989, Plant Mol. Biol. 14:51).Wounding reportedly results in the rapid accumulation of proteinaseinhibitors not only in wounded leaves but also in distal, unwoundedleaves, suggesting that a signal, or signals, released from the woundsite travels throughout the plant. Transport of these signals ismediated locally through intercellular and intracellular fluids thatpermeate wound or infection sites (Green, T. R. and C. A. Ryan, Science35 175:776-777, 1972) or travel systemically through the vascular systemof plants (Kuc, J. and C. Presisig, Mycologia 76:767-784,1984: M. Kopp,et al., Plant Physiol. 90:208-216, 1990; and K. E. Hammond-Kosack, etal., Physiol. Mol Plant Path. 35:495-506, 1989). Proposed wound signalsinclude: pectic fragments derived from the plant cell wall (C. A. Ryanand E. E. Farmer, 1991, Annu. Rev. Plant. Physiol. Mol. Bio. 42:651);the lipid-derived molecule, jasmonic acid (E. E. Farmer and C. A. Ryan,1990, Proc. Natl. Acad. Sci. U.S.A. 87:7713); the plant hormone,abscisic acid (H. Pena-Cortes, J. J. Sanchez-Serrano, R. Mertens, L.Willmitzer, S. Prat, 1989, Proc. Natl. Acad. Sci. U.S.A. 86:9851);electrical potentials (E. Davies, 1987, Plant, Cell and Environ. 10:623;J. F. Thain, H. M. Doherty, D. J. Bowles, D. C. Wildon, 1990, Plant,Cell and Environ. 13:569); and, more recently, an 18-amino acidpolypeptide called systemin (G. Pearce, D. Strydom, S. Johnson, C. A.Ryan, 1991, Science 253:895).

SUMMARY OF THE INVENTION

Disclosed herein are a) the isolation and sequencing of Systemin, an18-amino acid polypeptide (SEQ ID No.3) and first polypeptide hormonefound in plants; b) the isolation and sequencing of prosystemin, aprecursor 200 amino acid 23kDa polypeptide(SEQ ID No. 1); c) themolecular cloning of cDNA encoding prosystemin (SEQ ID No. 2) andgenomic DNA encoding prosystemin mRNA(SEQ ID No. 4); d) the constructionof antisense vectors encoding antisense RNA inhibiting prosysteminsynthesis; e) the construction of vectors containing the proseptemin orsystemin sense nucleic acid, as well as, f) a method of enhancing thedefense mechanism of plants. Systemin has been shown to be a powerfulinducer of the synthesis of wound-inducible plant defense proteinsincluding members of proteinase inhibitor families, i.e., the InhibitorI(8100 Da) and Inhibitor II(12,300 Da) families. Radioactively labelledsystemin applied to a plant wound site is rapidly translocated to distaltissues where it induces synthesis of defense proteins. Systemin isrepresented only once in the precursor prosystemin molecule and islocated close to the carboxy terminus of the precursor protein. Plantsexpressing antisense prosystemin RNA exhibit a greatly reduced synthesisof wound-induced proteinase inhibitors. Transgenic plants expressingsense prosystemin RNA exhibit an increased level of wound-inducedproteinase inhibitors. Manduca sexta larvae exhibit lowered growthweight when feeding on transgenic sense tomato plants compared to thoselarvae feeding on wild type plants.

Nucleic acid sequences of the invention are capable of encoding asystemin or prosystemin polypeptide or antisense RNA. The nucleic acidscomprise a nucleotide sequence capable of hybridizing with the sense orantisense strand of the nucleotide sequence of the prosystemin cDNA (SEQID No. 2) or genomic DNA(SEQ ID No. 4). The nucleic acids of theinvention encode prosystemin and systemin polypeptides or are antisensesequences which interfere with the expression of systemin or prosysteminin vivo. Systemin related polypeptides of the present invention comprisethe amino acid sequence R₁ R₁ QR₁ R₂ PPR₁ R₂ R₂ R₁ PPR₂ R₁ QR₁ R₁,wherein R₁ is any amino acid, R₂ is lysine or arginine (or anyderivative thereof, Q is glutamine (or any derivative thereof), and P isproline (or any derivative thereof. A representative example of asystemin polypeptide of the invention is the amino acid sequence: NH₃-AVQSKPPSKRDPPKMQTD-COO-(SEQ ID No. 3).

The processes of the present invention are useful for enhancingsynthesis of defense proteins in a plant by introducing a prosysteminsense nucleic acid into a plant cell, or inhibiting synthesis byintroducing an antisense nucleic acid. Transgenic plants containing thesubject nucleic acids of the invention are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of thisinvention will become more readily appreciated as the same becomesbetter understood by reference to the following detailed description,when taken in conjunction with the accompanying drawings, wherein:

FIG. 1 shows preliminary purification of systemin from the extracts oftomato leaves by semipreparative reverse-phase HPLC as described inExample 1, below.

FIG. 2 shows substantial purification of systemin by chromatography onan SCX-HPLC column as described in Example 1, below.

FIG. 3 shows the amino acid sequence of the systemin polypeptide(SEQ IDNo. 3).

FIG. 4 shows induction of defense protein synthesis, i.e., Inhibitor I(closed circles) and Inhibitor II (open circles), in tomato plants by asynthetic systemin polypeptide, as described in Example 2, below.

FIG. 5A shows the autoradiograph of a tomato leaf that was treated with¹⁴ C-labeled synthetic systemin polypeptide to demonstrate transport ofsystemin from wound sites into distal plant tissues.

FIG. 5B shows the ¹⁴ C-labeled synthetic systemin isolated byreverse-phase HPLC from the distal plant tissues of FIG. 5A.

FIG. 6 shows the amino acid sequence of prosystemin(SEQ ID No. 1).

FIGS. 7A and 7B show the nucleotide sequence of cDNA encodingprosystemin (SEQ ID No. 2) with crosshatched underlining showingrepeated sequence motifs and vertical bar underlining showing thelocation of systemin in the precursor sequence.

FIGS. 8A1 and 8A2 show the nucleotide sequence of the prosystemin genefrom position 1 to position 2100 (SEQ ID No. 4).

FIGS. 8B1 and 8B2 show the nucleotide sequence of the prosystemin genefrom position 2101 to position 4200 (SEQ ID No. 4).

FIG. 8C shows the nucleotide sequence of the prosystemin gene fromposition 4201 to position 4526 (SEQ ID No. 4).

FIG. 9A shows the organization of the prosystemin gene. The geneconsists of a 104 bp 5'-untranslated region, a 4176 bp coding regioncomposed of 11 exons (vertical bars) interrupted by 10 introns, and a246 bp 3'-untranslated region. The position of systemin is indicated bya horizontal bar labelled SYS.

FIG. 9B shows a Southern blot analysis of the prosystemin gene. Tomatogenomic DNA was isolated from leaves (as described in Example 6, below),and 5 μg was digested with EcoRI (lane 1); Bg1 II (lane 2) or Sca I(lane 3), and electrophoresed on a 0.8% agarose gel that was probed withnick-translated prosystemin.

FIG. 9C shows a Southern blot analysis of the species distribution ofprosystemin gene homologues, as described in Example 9, below. GenomicDNA (5 μg) from tomato (lane 1), potato (lane 2), tobacco (lane 3),alfalfa (lane 4), and Arabidopsis (lane 5) was digested with EcoRI andelectrophoresed on a 0.8% agarose gel. The gel was blotted ontonitrocellulose and probed with nick-translated prosystemin cDNA.

FIG. 10 shows the organization of the prosystemin gene. Exons arerepresented by vertical bars and numbered 1 to 11. The five exon pairsare: 1 plus 2; 3 plus 4; 5 plus 6; 7 plus 8 and 9 plus 10.

FIG. 11 shows sequence alignment of the prosystemin gene exons. Theconsensus sequence (con) is composed of those bases that occur at thesame position in at least three of the five exon sequences.

FIGS. 11A shows the alignment of sequences of the first exons of eachpair(exon 3, bases 1285-1373 of SEQ ID No. 4; exon 7, bases 2442-2505 ofSEQ ID No. 4; exon 5, bases 2051-2117 of SEQ ID No. 4; exon 9, bases3352-3400 of SEQ ID No. 4; exon 1, bases 105-138 of SEQ ID No. 4; and,con, SEQ ID No. 5).

FIGS. 11B shows the alignment of the sequences of the second exons ofeach pair (exon 4, bases 1483-1522 of SEQ ID No. 4; exon 8, bases2672-2709 of SEQ ID No. 4; exon 6, bases 2275-2318 of SEQ ID No. 4; exon10, bases 3545-3582 of SEQ ID No. 4; exon 2, bases 286-332 of SEQ ID No.4; and, con, SEQ ID No. 6) .

FIG. 12 shows sequence alignment of three repeated polypeptide sequenceswithin prosystemin (SEQ ID No.1). Three polypeptide sequences (Rep A,Rep B and Rep C; each occurring once within the amino-terminal half ofprosystemin), are aligned with the homologous sequences (Rep 2A, Rep 2Band Rep 2C; each occurring once within the carboxy terminal half ofprosystemin). Amino acids which differ between repeats are underlined.The amino acids at the beginning and end of each repeat are numberedfrom the amino terminus of prosystemin.

FIG. 13A shows the positions of the duplicated polypeptide sequenceswithin prosystemin. Prosystemin is represented by a horizontal bar withthe amino acid residues numbered 1 to 200 from the amino terminus.Sequence elements Rep A, Rep B and Rep C and their repeats Rep 2A, Rep2B and Rep 2C are indicated by hatched bars. Systemin is represented bya hatched bar labelled Sys.

FIG. 13B shows the location of the sequences encoding the polypeptiderepeats within the prosystemin gene. Exons are represented by verticalbars. The parts of the exons encoding the polypeptide repeats areshaded.

FIG. 14 shows a sequence comparison of the intron boundary at the 3'-endof the exons 3 (SEQ ID No.7) and 7 (SEQ ID No. 8). Exon sequence isunderlined. The first four bases of the intron at the 3'-end of exon 7have been displaced to facilitate accurate alignment of the homologoussequences occurring at the 3'-end of exon 3 and at the 5'-end of theintron between exons 7 and 8.

FIG. 15A shows a Northern blot analysis of the time course of inductionof prosystemin mRNA and Inhibitor I mRNA after wounding, as described inExample 7, below.

FIG. 15B shows a Northern blot analysis of the distribution ofprosystemin mRNA in various parts of an unwounded, fully-grown tomatoplant, as described in Example 8, below. Total RNA was extracted fromthe following parts of an unwounded tomato plant; root (R); stem (St);petiole (Pt); leaf (Le); sepal (Se); petal (Pe); stamen (Sm) and pistil(Pi).

FIG. 16A shows a Northern blot analysis of total RNA extracted fromtransgenic antisense plant 1A4. Lane 1 shows the results obtained withthe sense probe and Lane 2 shows the results with the antisense probe,as described in Example 10, below.

FIG. 16B shows a graphic depiction of the levels of Inhibitor I inwounded F1 transgenic antisense plants (unshaded bars) andnon-transformed control plants (solid bars), as described in Example 10,below.

FIG. 16C shows a graphical depiction of the levels of Inhibitor II inwounded F1 transgenic antisense plants (unshaded bars) andnon-transformed control plants (solid bars), as described in Example 10,below.

FIG. 17 shows a Northern blot analysis of total RNA extracts collectedat different times from undamaged leaves of control (nontransformed) andtransgenic tomato plants during the feeding experiments with Manducasexta larvae, as described in Example 11, below.

FIG. 18 shows the time course of accumulation of Inhibitor I and 11proteins in undamaged leaves of control and transgenic tomato plantsinduced by the feeding of Manduca sexta larvae, as described in Example11, below.

FIG. 19 shows the growth of Manduca sexta larvae, while feeding onleaves of control and transgenic antisense tomato plants, as describedin Example 11, below.

FIG. 20 shows the time course of larval weight gain of Manduca sextalarvae feeding on leaves of wild type and transgenic tomato plants, asdescribed in Example 11, below.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

As used herein the following terms are used to mean:

The term "defense proteins" is intended to include proteins that impedeplant tissue attack or ingestion by predators, such as by herbivores,insects, fungi, bacteria or viruses. Defense proteins increaseresistance of plants to predator attack by acting directly to impedeplant tissue attack or ingestion, or by acting indirectly to produceother defense compounds from precursor materials, (e.g., by acting toinduce enzymes in a pathway synthesizing defense compounds; or, byinducing proteins that regulate enzymes that synthesize defensecompounds). Representative examples of defense proteins include: e.g.,proteinase inhibitors, thionins, chitinases and β-glucanases.Representative enzymes that lead to the synthesis of defense compoundsinclude, e.g., casbene synthase. Representative enzymes that are part ofa biosynthetic pathway leading to synthesis of defense compoundsincludes, e.g. enzymes in the phenylpropenoid and terpenoid pathways forsynthesis of phytoalexin antibiotics, alkaloids and other toxicchemicals. Other predator defense proteins useful in connection with theinvention disclosed herein will, of course, be apparent to those skilledin the art. Particularly suitable predator defense proteins includeinhibitors of digestive proteolytic enzymes of the attacking herbivore,such as proteinase inhibitors, and antibacterial, antimycotic, andantiviral compounds and the like. Representative proteinase inhibitordefense proteins include, e.g., the Kunitz family of trypsin inhibitors,the Bowman-Birk family of proteinase inhibitors, the Inhibitor I familyof proteinase inhibitors, the Inhibitor II family of proteinaseinhibitors, the barley family of trypsin inhibitors, and the squashfamily of proteinase inhibitors. Representative examples of plantproteinase inhibitors are disclosed in PCT/US/91/03685, acontinuation-in-part application of U.S. patent application Ser. No.07/528,956, the disclosures of both applications are incorporated hereinby reference.

The term "nucleic acid" is intended to mean natural and synthetic linearand sequential arrays of nucleotides and nucleosides, e.g., in cDNA,genomic DNA (gDNA), mRNA, and RNA, oligonucleotides, oligonucleosides,and derivatives thereof. For ease of discussion, such nucleic acids areat times collectively referred to herein as "constructs," "plasmids," or"vectors." Representative examples of the nucleic acids of the inventioninclude bacterial plasmid vectors such as expression, cosmid, andcloning and transformation vectors (e.g., pBR322, λ, Ti, and the like),plant viral vectors (e.g., modified CaMV and the like), and syntheticoligonucleotide molecules such as chemically synthesized RNA or DNA.

The term "encoding" is intended to mean that the subject nucleic acidmay be transcribed and translated into the subject protein in a cell,e.g., when the subject nucleic acid is linked to appropriate controlsequences such as promoter and enhancer elements in a suitable vector(e.g., an expression vector) and when the vector is introduced into acell.

The term "polypeptide" is used to mean three or more amino acids linkedin a serial array.

The term "antisense DNA" is used to mean a gene sequence DNA that has anucleotide sequence homologous with the "sense strand" of a gene whenread in a reversed orientation, i.e., DNA read into RNA in a 3' to 5'rather than 5' to 3' direction. The term "antisense RNA" is used to meana RNA nucleotide sequence (e.g., encoded by an antisense DNA orsynthesized complementary with said antisense DNA). Antisense RNA iscapable of hybridizing under stringent conditions with an antisense DNA.The antisense RNA of the invention is useful for inhibiting expressionof a "target gene" either at the transcriptional or translational level.For example, transcription of the subject nucleic acids may produceantisense transcripts that are capable of inhibiting transcription byinhibiting initiation of transcription or by competing for limitingtranscription factors; or, the antisense transcripts may inhibittransport of the "target RNA"; or, the antisense transcripts may inhibittranslation of "target RNA".

The term "sense strand" is used to mean the single stranded DNA moleculefrom a genomic DNA that is transcribable and translatable into thepolypeptide product of the gene. The term "antisense strand" is used tomean the single strand DNA molecule of a genomic DNA that iscomplementary with the sense strand of the gene.

The term "capable of hybridizing under stringent conditions" is used tomean annealing a first nucleic acid to a second nucleic acid understringent conditions (defined below). For example, the first nucleicacid may be a test sample, and the second nucleic acid may be the senseor antisense strand of a prosystemin gene. Hybridization of the firstand second nucleic acids is conducted under stringent conditions, e.g.,high temperature and/or low salt content, which tend to disfavorhybridization of dissimilar nucleotide sequences. A suitable protocolinvolving hybridization in 6 X SSC, at 42° C. in aqueous solutionfollowed by washing with 1 X SSC, at 55° C. in aqueous solution isprovided in the illustrative examples below. (Other experimentalconditions for controlling stringency are described in Maniatis, T., etal., Molecular Cloning; A Laboratory Manual, Cold Springs HarborLaboratory, Cold Springs, N.Y., 1982, at pages 387-389; and also inSambrook, Fritsch, and Maniatis, Molecular Cloning; A Laboratory Manual,Second Edition, Volume 2, Cold Springs Harbor Laboratory, Cold Springs,N.Y., 1989, pages 8.46-8.47.)

The term "fragment" when used herein with reference to nucleic acid(e.g., cDNA, genomic DNA, i.e., gDNA) is used to mean a portion of thesubject nucleic acid such as constructed artificially (e.g., throughchemical synthesis) or by cleaving a natural product into a multiplicityof pieces (e.g., with a nuclease or endonuclease to obtain restrictionfragments).

The term "synthetic oligonucleotide" refers to an artificial nucleicacid (e.g., a chemically synthesized nucleic acid) having 9 or morenucleotides.

The term "systemin polypeptide" is used to mean a polypeptide having anamino acid sequence R₁ R₁ QR₁ R₂ PPR₁ R₂ R₂ R₁ PPR₂ R₁ QR₁ R₁, whereinR₁ is any amino acid, R₂ is lysine or arginine (or derivative thereof, Qis glutamine (or derivative thereof), and P is proline (or any otherderivative thereof), e.g., the systemin polypeptide of FIG. 3; namely,NH₃ -AVQSKPPSKRDPPKMQTD-COO- (SEQ ID No. 3). Skilled artisans willrecognize that through the process of mutation and/or evolution,polypeptides of different lengths, e.g., with insertions, substitutions,deletions, and the like, may have arisen that are related to thesystemin polypeptide of the invention by virtue of: a) amino acid and/ornucleotide sequence homology; b) a defensive function in regulating geneexpression in response to predators, pathogens, and mechanical injury;and/or, c) the organization of the genomic DNA, as described in Example6, below. Representative examples of systemin family members in tomatoand potato are provided in Example 6-9 (below), and illustrative methodsfor identification of systemin family members in other species, genra,and families of plants are also provided in Examples 6-9 (below).

The term "systemin nucleic acid" is used herein to refer to that subsetof nucleic acids capable of encoding a systemin polypeptide.

The term "prosystemin polypeptide" is used to mean a precursorpolypeptide capable of giving rise to a systemin polypeptide. Arepresentative example is provided by the prosystemin polypeptideencoded by the cDNA of FIGS. 7A and 7B (SEQ ID No. 2) or the codingregion of the genomic DNA of FIGS. 8A1, 8A2, 8B1, 8B2 and 8C (SEQ ID No.4). Prosystemin polypeptide is capable of being cleaved (e.g.,chemically or enzymatically) to give rise to systemin. A representativemethod for identifying prosystemin genes in different species of plantsis provided in Example 9, below.

The term "prosystemin nucleic acid" is used herein to refer to thatsubset of nucleic acids capable of encoding a prosystemin polypeptide.

Embodiments of the invention described and illustrated below providesystemin and prosystemin polypeptides, nucleic acids encoding systeminand prosystemin mRNA, cDNA, and genomic DNA, and, including 5'regulatory sequences controlling transcription of prosystemin gDNA intomRNA. The subject nucleic acids of the invention are capable of encodingprosystemins that are constitutively synthesized at a low level andwound-inducible to a high level (see illustrative Example 7, below).Would-inducible and constitutive low-level expression is provided byregulatory elements within 3000 bp of the 5' region of the systemin genesequence, the first 104 nucleotides of which are shown in FIG. 8A (SEQID No. 4). Promoter, enhancer, and other regulatory elements within the3000 bp 5' region are useful for insertion into recombinant plasmids andvectors for controlling gene expression in plants, (i.e., genes otherthan prosystemin). Representative examples of genes that may be linkedto the 5' regulatory elements of prosystemin include; genes encodingstorage or nutritionally important proteins, such as vegetative storageproteins, seed storage proteins, tuber storage proteins and the like;and, genes encoding other plant defense genes, i.e., other proteinaseinhibitors Bt toxen, and the like; genes encoding regulatory enzymes formetabolic and defensive processes, including phenylalanine amines, HMGCIA reductase and the like; genes encoding commercially importantenzymes in plant syspension culture, such as proteinases, lipases, andthe like; and, genes that regulate flower color.

Purification and physical properties of a representative systeminpolypeptide are disclosed (Example 1, below). Skilled artisans willrecognize that the relatively high proportion of hydrophilic amino acidsin the prosystemin polypeptide suggest a variety of conventionalapproaches to purification that may be used to purify a natural,recombinant, or synthetic prosystemin polypeptide, (e.g., ion exchangechromatography, affinity chromatography, specific ion precipitation, andthe like).

The subject amino acid sequence of prosystemin disclosed herein providesamino acid sequences that may be used to construct synthetic peptides ofprosystemin or systemin; or, alternatively they may be used to instructsites at which cleavage of a prosystemin polypeptide will liberate asystemin (e.g., enzymatic cleavage sites in a natural prosystemin or achimeric recombinant prosystemin protein. In the latter case a chimericrecombinant prosystemin polypeptide may be produced in an expressionsystem, the chimeric protein purified, and then systemin liberated fromthe chimeric protein by enzymatic cleavage.). Cleaving a prosysteminpolypeptide at boundary amino acids produces systemin, e.g., cleavingthe prosystemin of FIG. 7B (SEQ ID No. 2) at both Leu₁₇₈ -Ala₁₇₉, (e.g.,cleaving with an Leu-Ala-specific endopeptidase; abbreviated, LApeptidase) and at Asp₁₉₆ -Asn₁₉₇ (e.g., cleaving with anAsp-Asn-specific endopeptidase; abbreviated, DN peptidase). As analternative to the LA peptidase, a prosystemin polypeptide may also becleaved by suitable enzymes at other upstream sites such as Arg₁₇₅-Glu₁₇₆ or Glu₁₇₆ -Asp₁₇₇ ; followed by sequential cleavage of theproduct with an N-terminal peptidase, i.e., until the LA residues arereached and cleaved. In a similar manner and as an alternative to a DNpeptidase, a carboxypeptidase or carboxydipeptidase may be used tosequentially remote amino acids, from the carboxy-terminus until the DNresidues are reached and cleaved. Those skilled in the art willrecognize that a suitable LA-specific peptidase(s) may be isolated fromplant tissues, e.g., by using natural (or synthetic) polypeptidesubstrates having the prosystemin-systemin boundary amino acid sequences(e.g., L-A and D-N) and assaying for the production of systeminbiological activity. In one such illustrative example, a recombinantprosystemin chimeric protein may be synthesized by an expression systemand used as a substrate in enzymatic assays to identify and isolate theLA and/or DN peptidase(s).

Those skilled in the art will recognize that the subject prosysteminamino acid sequence may be used for constructing proteinase inhibitorsspecific for the LA and/or DN peptidases, and such inhibitors may beuseful for inhibiting systemin production from prosystemin; therebyinhibiting systemin activation of defense protein production in plants.Skilled artisans will also recognize that LA and DN peptidase may beselected with enhanced ability to liberate systemin from prosystemin(e.g. LA and DN enzymes having increased turnover number, decreased Km,increased Vmax, or decreased sensitivity to feedback inhibition, and thelike). Strains of plants may either be selected, or constructed (i.e.,as transgenic plants), having increased LA and/or DN peptidase activity.The subject plants may exhibit increased resistance to predators.

The subject systemin polypeptides of the present invention may also beused for identifying and isolating systemin receptors from plant cells.Those skilled in the art will recognize that the subject polypeptidescan be labeled (e.g., with a radioactive label) and conjugated to aphotochemical crosslinking agent. The subject conjugated andradiolabeled polypeptides bind to the cellular systemin receptor andphotochemical activation forms covalent bonds between the polypeptideand its receptor. When the receptor-polypeptide complex is extractedfrom the cell it may be isolated and identified by virtue of its label,e.g., the molecular size may be conveniently determined by SDS-PAGE andautoradiography. The subject polypeptides of the invention may also beuseful in ligand affinity chromatography for isolating systeminreceptors.

Embodiments of the invention provide processes for enhancing orinhibiting synthesis of a defense protein in a plant by introducing thesubject nucleic acids of the invention into a plant cell. In onerepresentative example enhanced defense protein production may beachieved by inserting prosystemin (or systemin) nucleic acid in a vectordownstream from a promoter sequence capable of driving constitutivehigh-level expression in a plant cell. In the latter case, when thesubject vector is introduced into a plant cell the cells containing oneor more copies of the subject nucleic acid may exhibit increasedsynthesis of systemin. When grown into plants the transgenic plants mayexhibit increased synthesis of defense proteins, and increasedresistance to herbivores, as discussed in more detail in Example 11,below.

In another embodiment, the invention provides processes for inhibitingsynthesis of defense proteins in a plant by inserting prosysteminantisense nucleic acid in a vector downstream from a promoter sequence.When the latter construct is introduced into plant cells the cellscontaining one or more copies of the subject nucleic acid may exhibitdecreased synthesis of defense proteins. A representative example of aprosystemin antisense vector, and process for inhibiting synthesis ofdefense proteins is provided in Example 10, below.

Transgenic plants containing the subject antisense nucleic acids of theinvention are useful for: a) identifying other mediators that may bepresent in the prosystemin molecule, (e.g., other mediators that mayinduce expression of defense proteins or differentiation); b)establishing the extent to which any specific insect and/or pathogen isresponsible for damage of a particular plant. In the latter case thetransgenic plants of the invention are useful for assessing theimportance of systemin defense mechanisms in production of a plant as acrop.

In still other embodiments, the invention provides transgenic plantsconstructed by introducing a subject nucleic acid of the invention intoa plant cell, and growing the cell into a callus and then into a plant;or, alternatively by breeding a transgenic plant from the subjectprocess with a second plant to form an F1 or higher hybrid (i.e., F2).The subject transgenic plants and progeny may be used to find thoseplants that contain extra copies of the subject nucleic acid of theinvention, and increased expression of prosystemin or systemin. Arepresentative example of a process for producing such a transgenicplant, and breeding it to obtain F1 offspring is provided below inExample 10.

Those skilled in the art will recognize the agricultural advantagesinherent in plants constructed to have either increased or decreasedexpression of systemin polypeptide. For example, such plants may haveincreased resistance to attack by predators, insects, pathogens,microorganisms, herbivores, mechanical damage and the like. Skilledartisans will also recognize that chemical agents may be developed thatwill mimic or induce systemin activity (e.g., in a manner similar tomethyl jasmonate induction of systemin activity), and these chemicalagents may be useful when sprayed on plants in maximizing cropresistance to herbivores, pathogens, and mechanical damage.Representative examples of plants in which the process may be usefulinclude (but are not limited to) tomato, potato, tobacco, corn, wheat,rice, cotton, soybean, alfalfa, rape, poplar trees, pine and fir treesand the like.

The subject nucleic acids of the invention are also useful asoligonucleotide probes (e.g., ³² P-labeled synthetic oligonucleotidesconstructed to complement portions of prosystemin nucleotide sequence),and restriction fragment probes (e.g., end-labeled restriction fragmentsof prosystemin cDNA), in Northern and Southern blots for selecting andscreening among plants to find natural and mutant strains with increasedprosystemin expression and/or genomic copy number. This screeningprocedure can be useful for identifying plant strains with increasedresistance to attack by predators, herbivores, insects, bacteria, fungi,viruses, mechanical damage, and the like.

The subject polypeptides of the invention are useful for inducingmonoclonal and polyclonal antibodies that may be used in immunoassays todetect the presence or amount of a prosystemin or systemin polypeptidein plant tissues, extracts, and fluids (e.g. see E. Harlow and D. Lane,Antibodies: A Laboratory Manual, Cold Springs Harbor Laboratory, ColdSprings, N.Y. 1988). The latter immunoassays may prove useful foridentifying natural and mutant strains of plants with increased levelsof prosystemin or systemin. Strains exhibiting increased levels of thesubject polypeptides may have increased resistance to attack byherbivores, i.e., insects, bacteria, fungi, and viruses.

Systemin is a primary polypeptide signal mediating wound-inducibleexpression of defense genes in distal leaves and, therefore, is thefirst example of a peptide hormone found in plants. The expression of arecombinant antisense genetic expression construct (i.e., containing anantisense prosystemin cDNA) resulted in an almost complete suppressionof systemic wound induced defense protein synthesis in plants. Thelatter finding provides evidence that systemin is an integral componentof the systemic signal transduction system in plants that inducesdefense protein synthesis in response to attack by predators and thelike. It is considered most likely that systemin is the first member tobe identified in a systemin family of plant polypeptide hormones. It isconsidered highly likely that members of the systemin family mayregulate developmental events in the meristems, flower tissues, andfruit of plants, e.g., tomatoes and potatoes. Other members of thesystemin family may be identified by virtue of their amino acid ornucleotide sequence homology with prosystemin or systemin, or by theirability to hybridize with the subject prosystemin or systemin nucleicacids of the invention. (In this regard, the nucleotide sequences of theexons identified in Example 6, below, may prove useful asoligonucleotide probes for identifying other systemin family members.)In this case the ability of a DNA or RNA to hybridize with the nucleicacid of the invention under conditions of reduced stringency, (e.g., asuitable protocol involving hybridization in 6 X SSC, at 42° C. inaqueous solution followed by washing with 1 X SSC, at 55° C. in aqueoussolution) will be considered a preliminary indication that the DNA orRNA contains a systemin family member. The DNA or RNA may then besequenced and the sequence compared with the sequence of systemin andprosystemin. (Experimental conditions for controlling stringency arealso described in Maniatis, T., et al., Molecular Cloning; A LaboratoryManual, Cold Springs Harbor Laboratory, Cold Springs, N.Y., 1982, atpages 387-389; and, also in Sambrook, Fritsch, and Maniatis, MolecularCloning; A Laboratory Manual, Second Edition, Volume 2, Cold SpringsHarbor Laboratory, Cold Springs, N.Y., 1989, pages 8.46-8.47.) Systeminfamily members may be recognized by virtue of about 50% to about 100%,or more preferably about 70% to about 100%, and most preferably about80% to about 100% homology at the amino acid or nucleotide level, i.e.,over a stretch of about 5 or more amino acids or about 15 or morenucleotides.

The foregoing may be appreciated more fully by reference to thefollowing representative examples of the subject compositions andmethods provided by the invention.

EXAMPLE 1 Isolation and Sequencing of the Systemin Polypeptide

Oligogalacturonides were initially considered to be primary candidatesas systemic signals for the wound response because they elicit synthesisof antibiotic phytoalexins in plant cells near the sites of infections(10, 11). Oligogalacturonides are released by pectin-degrading that arenot found in tomato leaves. In addition, when labeled α-1,4-oligogalacturonides were applied to wound sites on tomato plants, theywere not found to be mobile (E. A. -H. Baydoun and S. Fry, Planta 165,269 1985). Thus, oligogalacturonides are probably not involved assystemic mediators of signal transduction in plants, at least withrespect to induction of proteinase inhibitor genes in response towounding.

A search was initiated for systemic signals inducing proteinaseInhibitor I and II genes in tomato leaf extracts. This search led us toidentify a polypeptide in tomato leaves that is free of carbohydratesand induces proteinase inhibitor activity when supplied to young tomatoplants. The polypeptide was purified using high-performance liquidchromatography (HPLC, see Materials and Methods, below). Inducingactivity of the polypeptide was assayed by cutting the petioles of youngplants and introducing eluted fractions from column separations into thecut over a period of 30 min. The plants were subsequently transferred tosmall vials of water, incubated under constant light for 24 hours asdescribed (C. A. Ryan, Plant Physiol. 54, 328, 1974), and the amount ofproteinase Inhibitor I and II in the leaf juice was quantified by radialimmunodiffusion in agar gels that contained rabbit antiserum toInhibitor I or Inhibitor II (C. A. Ryan, Anal. Biochem. 19, 434, 1967;R. Trautman, K. Cowan, G. Wagner, Immunochemistry 8, 901, 1971). Over30,000 young tomato plants were assayed over a 2.5 year period. With theuse of this protocol, slightly more than 1 μg of an active factor (i.e.,systemin) was isolated from approximately 60 pounds of tomato leaves.

The elution profile of the preliminary extract of tomato leaves (FIG. 1)was complex. Several fractions exhibited proteinase inhibitor inducingactivity but one peak (FIG. 1) was selected for further purificationbecause it contained the highest activity and the best yield from thepurification.

After several additional purification steps (see Materials and Methods,below), a major peak that possessed high specific activity was elutedfrom a strong cation exchange (SCX) HPLC column (FIG. 2). The propertiesof the eluted material resembled those of a polypeptide, that is,absorbance in the spectral region appropriate for peptide bonds, totalloss of activity and recovery of free amino acids after acid hydrolysis,partial loss of activity in the presence of trypsin and otherproteolytic enzymes, and a positive assay result with bicinchoninic acid(P. K. Smith et al., Anal. Biochem. 150, 76, 1985). Total amino acidanalysis of the bioactive peak eluted from the SCX-HPLC (step 5,Materials and Methods, below) was determined as described below. Theamino acid sequence analysis of the active component (conducted asdescribed below; see, Materials and Methods) identified its length anddetermined the sequence of FIG. 3: NH₃ -AVQSKPPSKRDPPKMQTQTD-COO- (SEQID No. 3)). No significant similarities were found to known proteinsequences and the polypeptide was named "systemin" (ProteinIdentification Resource release 26; Pearson/Lipman FASTA program at theMolecular Biology Computer Research Resource, Harvard Medical School).The sequence is a palindrome: xxQxBPPxBBxPPBxQxx (x, any amino acidresidue; B, Lys or Arg; Q, Gln; P, Pro). A synthesized polypeptide ofidentical sequence to the systemin sequence (prepared as describedbelow; see, Materials and Methods) eluted from the C18 (step 2) columnwith the same retention time as the native polypeptide.

Materials and Methods:

Purification and isolation of the polypeptide inducer of defenseproteins:

Step 1: Approximately 2 kg of tomato leaves Lycopersicon esculentum (v.Castlemart) were harvested from 20-day-old plants, grown under cycles of17 hours light at 28° C. and 7 hours dark at 18° C. Leaves werehomogenized in a Waring blender for 5 min with distilled water (totalvolume of 4 liters) and filtered through four layers of cheesecloth. Theliquid was adjusted to pH 4.5 with HCI and centrifuged at 1000 g for 10min. The supernatant was adjusted to pH 6.1 with 10N NaOH, centrifugedat 10,000 g for 10 min at 20° C., and decanted through Whatman #4 filterpaper. The filtrate was chromatographed on DEAE cellulose, followed byreversed-phase C18 flash chromatography, Sephadex G25 gel filtration,and then CM Sephadex chromatography.

The DEAE cellulose column (Whatman DE52, 5.9 cm by 15 cm) wasequilibrated in 1M ammonium bicarbonate and washed exhaustively withdistilled water. The material eluting in the void volume was collectedand stored overnight at 4° C. TFA was added dropwise to the stored eluteto a final concentration of 0.2% (v/v); the solution was then clarifiedby centrifugation at 20,000 g for 5 min at room temperature. Thesupernatant was loaded onto a reversed-phase flash column (C18, 40 μm, 3cm by 25 cm) previously equilibrated with aqueous 0.1% TFA. The columnwas eluted with the use of compressed nitrogen at 8 psi. After thesample was loaded, the column was washed with 200 ml 0.1% TFA; theretained material was then eluted with successive washes of 20, 40, and60% methanol in 0.1% TFA. The methanol was removed with a rotaryevaporator and the remaining liquid was frozen and lyophilized. Twokilograms of leaf material yielded about 1 g of crude materialcontaining systemin. The procedure was repeated 15 times. Samples(approximately 4 g) of crude material dissolved in 20 ml water andadjusted to pH 7.8 with 10M ammonium hydroxide were loaded onto a G25Sephadex column (4 cm by 44 cm) that was equilibrated with 50 mMammonium bicarbonate, pH 7.8. The material eluting at and just after thevoid volume was recovered and lyophilized. Four identical runs throughthe entire procedure produced 1.25 g of partially purified systemin. The1.25 g was dissolved in 500 ml H₂ O, the pH was adjusted to 6 with 1MNaOH, and the sample was applied to a CM Sephadex column (2 cm by 17 cm)and washed with 0.01M potassium phosphate, pH 6. The activity wasretained by the CM Sephadex, eluted with 250 mM ammonium bicarbonate,and lyophilized. The total yield of proteins in this step was 190 mg.

Step 2: The active fraction (190 mg) recovered from step 1 was dissolvedin 10 ml 0.1% TFA, centrifuged at 20,000 g for 5 min, filtered, andchromatographed on a reversed-phase C18 column. Five microliters of each2-ml eluted fraction was diluted to 360 μl with 154 mM sodium phosphate,pH 6.5, and assayed for proteinase Inhibitor I inducing activity (x inFIG. 1). Four plants were assayed per fraction. The material wasinjected into a semi-preparative reversed-phase C18 column (Vydac,Hesperia, CA, Column 218 TP510, 10 mm by 250 mm, 5-μm beads, 300Apores). Solvent A consisted of 0.1% TFA in water. Solvent B consisted of0.1% TFA in acetonitrile. Samples were injected in solvent A and, after2 min, a 90 minute gradient to 30% solvent B was begun for elution. Theflow rate was 2 ml/min and eluted peaks were monitored at 225 nm.Several peaks of biological activity were found (as described below).The major peak of activity (shown in black in FIG. 1) resided in tubes43 to 46, which were pooled and lyophilized. Total protein content ofthe pooled factions was estimated at 2.5 mg.

Step 3: The total material recovered in step 2, above (2.5 mg), wassubjected to strong cation exchange HPLC on a poly-SULFO-ETHYLAspartamide (SCX) column (4.6 mm by 200 mm, 5 μm, The Nest Group,Southborough, Me.) with the use of the following solvent systems:Solvent A, 5 mM potassium phosphate, pH 3, in 25% acetronitrile; solventB, 5 mM potassium phosphate, 500 mM potassium chloride in 25%acetonitrile, pH 3. The sample was dissolved in 2 ml of solvent A,filtered, and applied to the column. After a 5-minute wash with solventA, a 60-min gradient to 50% B was applied. The flow rate was 1 ml/min,and the elution profile was monitored by absorbance at 210 nm. Theactive fractions, tubes 35 to 38, were pooled and reduced in volume to 1ml by vacuum centrifugation.

Step 4: The pooled fractions from step 3 were subjected to reverse-phaseC18 HPLC in 10 mM potassium phosphate, pH 6. Chromatography wasperformed on a Beckman Ultrasphere Ion pair column (4.6 mm by 250 mm,C18, 5 μm). Solvent A was 10 mM potassium phosphate, pH 6, and solvent Bwas 10 mM potassium phosphate, pH 6, containing 50% acetonitrile. Theactive fractions, tubes 39 to 42, were pooled and vacuum centrifuged toa final volume of 1 ml. This fraction was applied to the same column asthe previous run but under the solvent and gradient conditions of step2. The sample was adjusted to pH 3 with TFA, filtered through a 0.45-μmsyringe filter and chomatographed at a flow rate of 1 ml/min. The peaksof protein were detected at 212 nm. The fractions containing activity,eluting at 53.5 to 56.5 min, were pooled and vacuum centrifuged to avolume of 1 ml.

Step 5: The active fraction from step 4 was subjected to SCX-HPLC withthe same column and conditions as used in step 3, except that thegradient was shallower, i.e., the column was run at 0% B for 5 min atwhich time a gradient to 30% B in 120 min was started. Fractions (0.5ml) were diluted as in FIG. 1 and assayed for proteinase Inhibitor Iinducing activity (x in FIG. 2). The biologically active fractions ofthe system peak (shown in black in FIG. 2) were collected and analyzedfor amino acid content and sequence. The profile was detected byabsorbance at 210 nm. Fractions eluting at 76 to 78.5 min were pooledand vacuum centrifuged to reduce the volume to I ml.

Step 6: The step 5 fraction was desalted on a C18 HPLC column under theconditions of step 2. A 60-minute gradient to 30% solvent B wasemployed. The fractions containing the activity peak eluted at 55.0 to58.0 min and were pooled and concentrated by vacuum centrifugation to0.5 ml. The sample contained approximately 1 μg of protein, as estimatedby amino acid content after acid hydrolysis. The biological activity ofthe sample had the potential to induce maximal accumulation ofproteinase inhibitors in 40,000 tomato plants, (i.e., approximately40,000-fold purified). This sample was used for amino acid analysis andsequence determination.

Amino acid analysis:

The bioactive peak eluted from the SCX-HPLC column (step 6) was dried in6 by 50 mm glass tubes and hydrolyzed in the HCI vapor. The hydrolysateswere derivatized with phenylisothiocyanate and analyzed by reverse-phasechromatography on 30 cm by 0.39 cm columns (Picotag, Millipore)according to the manufacturer's suggestions.

Amino acid sequence analysis:

The amino acid sequence of the bioactive peak-eluted from the SCX-HPLCcolumn (step 6) was determined by established methods (D. J. Strydom etal., Biochemistry 25, 945, 1985; B. A. Bidlingmeyer, S. A. Cohen, T. L.Tarvin, J. Chromatogr. 336, 93, 1984). Briefly, sequencing was performedon a Beckman model 890 spinning cup instrument, equipped formicrosequencing, as recommended by the manufacturer, except that 0.1%water was added to the anhydrous heptafluorobutyric acid (HFBA) and 0.1%ethanethiol was added to the 25% trifluoracetic acid (TFA).Identification of the phenylthiohydantoin amino acids was byreverse-phase chromatography on an octadecylsilane column (IBM, Inc.; 30cm by 0.46 cm, 3-μm particle size) with the use of the gradient systemdescribed (D. J. Styrdom et al., supra). Abbreviations for the aminoacid residues are: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H,His; I, lie; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S,Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.

Synthesis of a synthetic systemin polypeptide:

A polypeptide corresponding to residues 2 through 18 (FIG. 3; SEQ ID No.3) was synthesized with the use of 9-fluorenylmethyl chloroformate(F-moc) solid phase chemistry with an Applied Biosystems Inc. Model 431Asynthesizer on a p-methyl benzyhyrylamine resin according to themanufacturer's protocol. The F-moc derivative of ¹⁴ C! Ala (New EnglandNuclear) was synthesized (J. Stewart and J. Young, Solid Phase PeptideSynthesis (Pierce Chemical Co., Rockford, Ill., ed. 2, 1984), pp.67-68)and added to the NH₂ -- terminal residue of the polypeptide; the ¹⁴ C!Ala-polypeptide was then cleaved from the resin. The radioactive peptidewas purified by C18 HPLC. The specific activity of the syntheticsystemin polypeptide was 19.0 μCi/μmol.

EXAMPLE 2 Systemic Translocation of Radiolabeled Systemin Polypeptide

The synthetic systemin polypeptide (described above) was tested forbioactivity and was found to be as effective as the native systeminpolypeptide (purified above to step 6) for inducing the synthesis andaccumulation of both Inhibitor I and II proteins when supplied to thecut stems of young tomato plants (FIG. 4). About 40 fmol of thepolypeptide per plant was required to produce half maximal accumulationof Inhibitors I and II, which represents about 10⁵ times more activityon a molar basis than the previously reported PIIF oligogalactunonideinducers derived from plant cell walls. The coordinate induction ofsynthesis of Inhibitor I and Inhibitor II proteins in response to thesystemin synthetic polypeptide (FIG. 4) is similar to a normal plantwound response that is transcriptionally regulated. This suggests thatthe polypeptide is activating the same signal transduction pathwayactivated by wounding (T. Green and C. A. Ryan, 1972, supra; J. S.Graham et al., 1986, supra), by oligosaccharides (P. Bishop et al.,1981, supra; M. Walker-Simmons et al., 1983, supra), and by methyljasmonate (E. E. Farmer et al., 1990, supra).

The systemin polypeptide, unlike the prior reported oligogalacturonides,is transported out of wounds to distal tissues. ¹⁴ C-labeled polypeptidewas synthesized (as described above, Example 1) and placed on freshwounds of tomato plants. Within 30 min the radioactivity had movedthroughout the leaf, and within 1 to 2 hours radiolabeled systemin wasidentified by HPLC (FIG. 5) in the phloem exudate (expressed from theplant according to the method described by R. W. King and J. A.ZeeVaart, Plant Physiol. 53, 96, 1974). The polypeptide was named"systemin" because of its mobility through phloem.

As well as being inducible by wounding in leaves, the genus forproteinase Inhibitors I and II are developmentally regulated in themeristems, flower tissues, and fruit of tomato species, and in potatotubers. Thus, it is likely that these developmental events may bemediated by systemin or by similar polypeptides that are members of thesystemin family.

FIG. 4 shows the results of experiments in which systemin syntheticpolypeptides inducted synthesis and accumulation of proteinase InhibitorI (•) and II (O) in the excised leaves of young tomato plants. Theleaves were incubated in solutions containing the synthetic systeminpolypeptide and the proteinase inhibitors were assayed as describedabove in Example 1. Each data point was obtained from assays of theleaves of 36 tomato plants.

EXAMPLE 3 Molecular Cloning Prosystemin

A prosystemin cDNA was isolated by screening a primary cDNA librarysynthesized from tomato leaf mRNA as follows:

Poly A+ mRNA was purified from tomato leaves using oligo dT columns(Pharmacia). cDNA was synthesized using the Stratagene cDNA synthesissystem and was cloned into lambda ZAP vector arms (Stratagene).Approximately 80,000 primary library recombinants were screened, byduplicate plaque lifts, using a degenerate oligonucleotide probe, termed"SP1", based on the amino acid sequence of the carboxy terminus ofsystemin (i.e. PPKMQTN; amino acids 190 to 196, as numbered in FIG. 6,excluding the last nucleotide residue of the Asp₁₉₆ codon). Thehybridization conditions for screening were: 6X SSC; 1X Denhardtssolution; 100 μg/ml, yeast tRNA; 0.5% sodium pyrophosphate andapproximately 4×10⁷ cpm of ³² P-end-labeled SP1. Hybridization was at37° C. for 36 hr. The filters were washed in several changes of 5X SSC,0.1% SDS at 48° C. for one hour. Approximately 50 positive clones wereidentified and rescreened using a second degenerate oligonucleotide,termed "SP2", corresponding to the amino terminus of systemin (AVQSKP;amino acids 179 to 184, as numbered in FIG. 6, excluding the last of thePro₁₈₄ codon). The hybridization and wash conditions were identical tothose used for SP1 except that the wash temperature for SP2 was 40° C.Of the initial positive clones only one hybridized to the SP2 probe.Fragments of the prosystemin cDNA, termed "pSYS 1", were subcloned intoBluescript® plasmids, single-stranded DNA was rescued and sequenced onboth strands by dideoxy sequencing using Sequenase (USB; Sanger et al.PNAS 73, 5463, 1977). Sequencing of the SP2 positive clone establishedthat it encoded the systemin polypeptide within the larger protein whichwas called "prosystemin". The prosystemin cDNA was not full-length,beginning at nucleotide 112 as numbered in FIG. 6 and SEQ ID No. 1.

The prosystemin cDNA (SEQ ID No. 2) consisted of 839 bp with an openreading frame encoding 197 amino acids. The reading frame remained opento the 5'-end of the clone, and since Northern blot analysis indicatedthat the systemin mRNA was 1 Kb in size, we concluded that the cDNA wasmissing approximately 100 bp at the 5'-end. The complete prosysteminmRNA sequence was subsequently determined by sequencing the prosystemingene (as described below in Example 6) and mapping the transcriptionalstart site (FIGS. 7A and 7B; Example 6). The experiments described inExample 6, below, established the length of the open reading frame as600 base pairs encoding a prosystemin protein of 200 amino acids. Theidentification of the initiating methionine codon was made on the basisof two criteria; the presence of multiple stop codons immediately 5' tothe methionine codon and the presence of an adjacent sequence similar tothe plant consensus sequence for translational initiation (H. A. Lutckeet al., 1987, EMBO Journal 6:43).

EXAMPLE 4 Structure and Properties of Prosystemin

Based on the cDNA sequence (FIGS. 7A and 7B; SEQ ID No. 2), systemin islocated close to the carboxy terminus of the 23 kDa prosystemin protein(FIGS. 7A and 7B; SEQ ID No. 2), amino acid residues 179 to 196;corresponding to nucleotides 639 to 699). The amino acid composition ofprosystemin is unusual in that it contains a high percentage of chargedamino acids; aspartic acid (10%), glutamic acid (17%), lysine (15%), butvery few hydrophobic amino acids. In consequence, prosystemin is amarkedly hydrophilic molecule. Analysis of the prosystemin sequencefailed to reveal a hydrophobic region at the amino terminus thatresembles a leader peptide. The post-translational processing pathwayand site of sub-cellular compartmentalization of prosystemin remain tobe determined. A search of the EMBL and GeneBank data base, with boththe cDNA and deduced protein precursor sequences failed to revealsignificant homology to any of the listed sequences.

Although the 18-amino acid systemin sequence occurs only once within theprecursor, close to the carboxy terminus, other sequence elements arerepeated. There is a short (6 to 9 amino acids), imperfect repeatoccurring five times within the prosystemin sequence (crosshatchedunderlining, FIGS. 7A and 7B). This observation suggests that at leastpart of the prosystemin gene may have evolved by multiple geneduplication/elongation events, a conclusion which is supported by thestructure of the gene.

EXAMPLE 5 Proteolytic Processing Sites in Prosystemin

The putative processing sites bordering systemin are shown in FIGS. 7Aand 7B; SEQ ID No. 2 (i.e. amino acid residues 178 and 197). The Leu(178) and Asn (197) processing sites do not conform to the consensussequence for the endoproteolytic processing sites flanking bioactivepeptides within animal prohormone precursors (e.g., see animal sites inR. B. Harris, 1989, Arch. Biochem. Biophys. 275(2):315 (1989). Theminimum animal consensus sequence consists of a pair of basic aminoacids which immediately precede the site of cleavage. In addition, thedibasic pair is often preceded, at a distance of two or three aminoacids, by a single basic amino acid. The animal consensus sequence is,however, found once within the prosystemin sequence, at residues 183-188(LysProProSerLysArg, FIGS. 7A and 7B; SEQ ID No. 2), which is a part ofthe mature systemin polypeptide. It is conceivable that the half-life ofsystemin is regulated by further processing at this site, e.g., to yieldan 8 amino acid carboxy-terminal peptide.

In animal systems prohormones are often processed to yield multiplebioactive peptides (J. Douglass, O. Civelli and E. Herbert, 1984, Ann.Rev. Biochem. 53:665; L. J. Jung and R. H. Schefler, 1991, Science251:1330) and members of the systemin family of plant polypeptidehormones may be subject to similar processing mechanisms.

EXAMPLE 6 Structure of the Prosystemin Gene and Systemin Gene Family

A primary library of 700,000 recombinants was plated on the bacterialstrain (P2) PLK-17 (Stratagene) and screened, by duplicate plaque lifts,with nick-translated prosystemin cDNA. Hybridization was carried out asdescribed below. A single positive clone was identified and purified.The gene was located on a 18 Kb genomic DNA fragment from which it wassub-cloned into Bluescript® plasmids. A series of overlapping, deletionsspanning most of the gene were produced using the Mung Bean/ExonucleaseIII system (Stratagene). Each deletion product was cloned into aBluescript® phagemid from which single-stranded DNA was rescued for useas the sequencing template. The gene was sequenced by the dideoxy methodof Sanger (supra) using Sequenase (U.S.B.). The sequence was completedusing custom-made oligonucleotide primers. The deduced sequence was thenconfirmed using custom-made oligonucleotide primers (as described in theMaterials and Methods below).

The sequence of the prosystemin gene is presented in FIGS. 8A1-8C andSEQ ID No. 4. The gene is composed of 4526 bp comprising a 104 bp5'-untranslated region, a 4176 bp coding region and a 246 bp3'-untranslated region. A striking feature of the sequence is that it is76% A:T-rich. The structure of the prosystemin gene is depicted in FIG.9A; Southern blot analysis is shown in FIG. 9B.

Within the prosystemin gene ten introns interrupt the coding region(FIG. 10). in consequence, the exons are small, ranging, in size from 34bp (exon 1) to 90 bp (exon 10). The first 10 exons are organized as fivepairs while the sequence encoding systemin is located on the final,unpaired exon. To investigate the relationship between the exon pairs wealigned the sequences of the first exon of each pair (exons 1, 3, 5, 7,9) and, separately, we aligned the sequences of the second exon of eachpair (exons 2, 4, 6, 8, 10), as shown in FIGS. 11A AND 11B (also SEQ IDNos. 4, 5 and 6). The sequences within the first exon group are allhomologous to each other as are the sequences within the second exongroup. There is no significant sequence homology between the two groups.These observations suggest that the five exon pairs arose by successiveduplications from a common ancestor. That the individual exons within apair are not homologous to each other suggests that the ancestral unitfrom which the gene is derived was a structure corresponding to the exonpair rather than to the individual exons within a pair.

In contrast to the set of five homologous exon pairs, the exon encodingsystemin (exon 11) does not show significant sequence homology with anyother part of the prosystemin gene. This observation suggests eitherthat the exon encoding systemin may have arisen separately from the restof the gene, to which it was subsequently added, or that the exonencoding systemin arose from the same ancestral sequence as the otherexons but subsequently evolved more rapidly.

Repeated amino acid sequences within prosystemin and the systemin genefamily.

The amino acid sequence of prosystemin, like that of the gene, is highlyrepetitive. A short oligopeptide sequence palendrome was identifiedabove in systemin (Example 1) and a similar theme occurring five timeswithin prosystemin that is encoded by the first exon of each of the fivehomologue pairs. In addition, the presence of three different,tandemly-repeated polypeptide elements within prosystemin provides aclue about the evolution of the prosystemin gene.

The tandemly repeated elements occur once within the amino-terminal halfof prosystemin and once within the carboxy-terminal half of prosystemin.The polypeptide elements within the amino-terminal half of the precursorhave been labelled Rep A, Rep B and Rep C and their repeats within thecarboxy-terminal half of the molecule have been labelled Rep 2A, Rep 2Band Rep 2C. The sequences of these repeated polypeptide elements areshown aligned in FIG. 12. The locations of the repeats withinprosystemin are shown in FIG. 13A, while the locations within theprosystemin gene of the DNA sequences encoding the polypeptide repeatsare shown in FIG. 13B. As can be seen from this comparison the Rep a,Rep B and Rep C polypeptides are encoded by two exon pairs (namely,three plus four and five plus six) while Rep 2A, Rep 2B and Rep 2C arealso encoded by two other exon pairs (namely, seven plus eight and nineplus ten).

The observations thus suggest that a set of polypeptide sequences,encoded by two pairs of exons, has been duplicated as one step in theevolution of the prosystemin gene. It would then appear that theancestral gene for prosystemin was subject to a tandem duplication of astructure composed of at least two pairs of exons. This conclusionprecludes a model in which the systemin gene evolved simply bysuccessive duplications of a single exon pair.

Intron boundaries may be shifted within repeated DNA sequences.

The amino acid sequences between the polypeptide Rep A, Rep B and Rep Cregions, that was found in the amino terminal half of prosystemin, werenot duplicated in the carboxy terminal half of the molecule whererepeats Rep 2A, Rep 2B and Rep 2C are almost contiguous (FIG. 13A). Thisobservation is largely explained by the fact that exon 7 (encodingelement Rep 2A), is truncated at its 3'-end (i.e., with respect to thesequence of exon 3, encoding element Rep A). Similarly, exon 9 (encodingelement Rep 2C), is truncated at its 5'-end (i.e., compared to exon 5,encoding element Rep C).

In the case of exon 7, the truncation did not arise by deleting a shortsection of the gene, but by shifting the position of the intron boundarywithin the existing sequence. Comparing the sequence around the intronjunction at the 3'-end of exon 7 with the corresponding junctionsequence at the 3'-end of exon 3 (FIG. 14; SEQ ID Nos. 6 and 7), it isevident that the sequence corresponding to the 3'-end of the exon 3constitutes the 5'-end of the intron between exons 7 and 8. Thisstructure may have arisen by either the elongation of exon 3 or theshortening of exon 7. It is not clear if the same process resulted inthe truncation of the 5'-end of exon 9.

Materials and Methods:

Primer extension was performed using a gel-purified oligonucleotideconsisting of bases 82 to 111 of the antisense strand of the cDNAsequence shown in FIGS. 7A and 7B (SEQ ID No. 2). Total RNA wasextracted from young tomato plants 4 hr after wounding and poly A+ mRNAwas isolated using oligo dT columns (Pharmacia). Three picomoles ofoligonucleotide were end-labelled using γ-ATP at a specific activity of6000/Ci/mmol. 2×10⁶ cpm of labelled oligonucleotide were annealed to 4μg polyA+ mRNA by heating to 85° C. for 10 min then hybridizingovernight at 30° C. in a solution of 40 mM PIPES, pH 6.4; 1 mM EDTA; 0.4M NaCl; 80% formamide. The annealed nucleic acids were ethanolprecipitated and resuspended in 30 μl of a solution containing: 50 mMTris, pH 7.5; 75 mM KCI; 10 mM dithiothreitol; 3 mM MgCl₂ ; 500 μM ofeach dNTP; 100 μg/ml bovine serum albumin. Thirty units of M-MLV ReverseTranscriptase (Stratagene) and one unit of RNase Block II (Stratagene)were added and the reaction mixture was incubated for 90 min at 37° C.At the end of the reaction one μl of 0.5M EDTA, pH 8.0 and one μl ofDNAse-free RNAse A (10 mg/ml) were added to the reaction mixture andincubated for a further 30 min at 37° C. The reaction mixture was phenolextracted, ethanol precipitated and resuspended in four μl TE buffer (10mM Tric-HCL, pH 7.5, 0.1 mM EDTA, pH 8.0) to which six μl of formamideloading buffer (80% formamide; 10 mM EDTA, pH 8.0; 1 mg/ml xylenecyanol; 1 mg/ml bromophenol blue) were subsequently added. Two μl of theresuspended products were analyzed on a 6% acrylamide/8M urea sequencinggel. The size standards were sequencing products generated using theprimer extension oligonucleotide as primer and single-stranded DNAderived from the 5'-end of the prosystemin gene as template. Sequencingwas carried out using Sequenase (USB) following the manufacturer'sinstructions for generating sequencing products close to the primer.

Mung Bean Nuclease analysis was carried out using a 400 bp Scal-Ndelfragment spanning the 5'-end of the prosystemin gene. The Ndel site islocated within the first exon of the systemin gene. The Ndel end of thefragment was end-labelled to a specific activity of 6×10⁶ cpm/μg andapproximately 10⁶ cpm were mixed with 4 μg of the same poly A+ RNA stockused in the primer extension experiment. The mixture was desiccated andresuspended in 15 μl of hybridization buffer. The mixture was coveredwith mineral oil, heated to 82° C. for 6 min then hybridized overnightat 37° C. The sample was then mixed with 200 μl of ice-cold Mung BeanNuclease buffer (30 mM sodium acetate (pH 5.0), 50 mM sodium chloride, ImM zinc chloride, 5% (v/v) glycerol) to which 10 units of Mung BeanNuclease (Stratagene) were added. The mixture was incubated for 30 minat 12° C. then extracted with an equal volume of a 1:1 mixture ofphenol:chloroform. The digestion products were coprecipitated with 1 μgof yeast tRNA and resuspended in 4 μl TE buffer plus 6 μl formamideloading buffer. Three μl of the resuspended digestion products wereanalyzed on a 6% acrylamide/8M urea gel. Size markers were generated byusing single-stranded DNA corresponding to the 5'-end of the gene astemplate. The sequencing primer was a 19-mer corresponding to the first19 bases (antisense strand) at the 3'-end of the Scal-Ndel probefragment.

EXAMPLE 7 Wound-Inducible Expression of the Prosystemin Gene

In considering the role of systemin as a mobile signal that activatesproteinase inhibitor genes in response to wounding, we investigated thepossibility that the prosystemin gene, itself, might be wound-inducible.Northern blot analysis was used to examine the levels of prosysteminmRNA and Inhibitor I mRNA in leaves of unwounded and wounded tomatoplants (FIG. 15A). Thirty-two young tomato plants were wounded threeweeks after germination. The plants had an upper and a lower leaf and asmall apial leaf. The lower leaf was wounded and mRNA was isolated fromthe upper (unwounded) leaf at the following time points after wounding:0.5, 1.5, 3, 6, 9, 12, and 24 hours. Four plants were used for each timepoint. Total RNA (5 μg) from each time point was electrophoresed on a1.4% agarose-formaldehyde gel and blotted onto nitrocellulose. The blotwas probed simultaneously with nick-translated prosystemin (SYS) andInhibitor I (Inh-1) cDNAs (see Materials and Methods, below).Prosystemin mRNA was found to accumulate in both wounded and unwoundedleaves of wounded tomato plants, demonstrating that prosystemin mRNA,like Inhibitor I mRNA, is systemically wound-inducible. Prosystemin mRNAreached the highest levels at three to four hours after wounding whileInhibitor I mRNA was most abundant eight to ten hours after wounding.Unlike the proteinase Inhibitor I message, which is absent in the leavesof unwounded tomato plants, a low level of prosystemin mRNA was detectedin the leaves of unwounded plants. Low, constitutive expression of theprosystemin gene in leaves may provide a continuous supply of systemin,allowing the plant to immediately respond to wounding.

The wound-induced accumulation of prosystemin mRNA and, presumably,prosystemin and systemin in the unwounded tissue may amplify the abilityof the plant to react to subsequent damage. Continued damage by insectattacks would, therefore, liberate more systemin from the newlysynthesized precursor than did the initial wounds, resulting in higherlevels of proteinase inhibitor synthesis as the attacks persist.

Since the initial rate of accumulation of prosystemin mRNA was fasterthan that of Inhibitor I mRNA in response to wounding (FIG. 15A), someaspects of the signal transduction pathways activating the two genes maydiffer. Additional signals may be responsible for the different rates ofaccumulation or the signal transduction pathways may respond to the samesignals but with different sensitivities.

Materials and Methods:

Nick-translation was performed using the NEN DuPont nick-translationsystem according to the manufacturer's instructions. Hybridization wascarried out under the following conditions: 50% formamide; 5X Denhardts;5X SSPE; 0.1% SDS; 100 μg/ml sheared salmon sperm DNA; 1 μg/ml poly Aand nick-translated DNA probe of specific activity approximately 10⁹cpm/μg. Unless otherwise stated, blots were washed in 1 X SSC, 0.1% SDSat 65° C.

EXAMPLE 8 Distribution of Prosystemin mRNA Throughout the Plant

Prosystemin mRNA is found throughout the aerial parts of the plant butnot in the roots (FIG. 15B). Total RNA was extracted from the followingparts of an unwounded, fully-grown tomato plant: root (R); stem (St);petiole (Pt), leaf (Le), sepal (Se), petal (Pe), stamen (Sm) and pistil(Pi)(FIG. 15B). Total RNA (5 μg) from each sample was electrophoresedand blotted as described in Example 7. The blot was probed withnick-translated prosystemin cDNA (as described in Example 7, above).

The highest constitutive levels of prosystemin mRNA are seen in theflower parts, a feature which is also characteristic of the distributionof Inhibitor I and Inhibitor II mRNAs. The general distribution ofprosystemin mRNA (at least in the parts of the plant above ground), isconsistent with the proposed role of systemin as a wound signal, sincewounding of any aerial part of the plant would be expected to result inthe systemic induction of proteinase inhibitor synthesis. The apparentabsence of prosystemin mRNA in the roots is surprising since we haveobserved the induction of proteinase inhibitor synthesis in tomatoleaves in response to wounding of the roots. It is possible that rootseither contain very low levels of prosystemin mRNA, undetectable in theassay, or they employ a different wound signal(s) (e.g., a differentsystemin gene family member) to activate proteinase inhibitor genes inthe leaves. It is also conceivable that prosystemin is transported fromleaves to roots where systemin is released in response to wounding.

EXAMPLE 9 Species Distribution of Prosystemin Gene Homologues

To determine if prosystemin gene homologues are found in other plantspecies, Southern and Northern blot analysis was performed on genomicDNA and total RNA from three species known to possess wound-inducibleproteinase inhibitors: potato, Solanum tuberosum, var. Russett Burbank(C. A. Ryan, 1968, Plant Physiol. 43, 1880), tobacco, Nicotiana tabacum,var. Xanthi (G. Pearce, results in preparation), and alfalfa, Medicagosativa, var. Vernema (W. E. Brown and C. A. Ryan, 1984, Biochemistry23:3418; W. E. Brown, K. Takio, K. Titani, C. A. Ryan, 1985,Biochemistry 24:2105); and, as a control, from one species (Arabidopsisthaliana, var. Columbia), which is not known to possess wound-inducibleproteinase inhibitors.

Southern blot analysis of the species distribution of prosystemin genehomologues is shown in FIG. 9C. Genomic DNA (5 μg) from tomato (FIG. 9C,lane 1), potato (FIG. 9C, lane 2), tobacco (FIG. 9C, lane 3), alfalfa(FIG. 9C, lane 4), and Arabidopsis (FIG. 9C, lane 5) was digested withEcoRI; restriction fragments were separated by electrophoresis on a 0.8%agarose gel; and, fragments of prosystemin were visualized by blottingto nitrocellulose and probing with nick-translated prosystemin cDNA. Theblot was washed at 55° C. under moderately stringent conditions. Of thefour plant species analyzed, a homologue of the prosystemin gene wasidentified under moderately stringent conditions only in potato (thenearest relative of the four species to tomato). A potato mRNA specieswas also identified that hybridized to tomato prosystemin cDNA and whichcomigrated with the tomato prosystemin mRNA. The nucleotide sequence ofthe gene in tobacco, alfalfa and Arabidopsis may have diverged from thatof the tomato gene to the extent that it can no longer be detected byhybridization under stringent conditions with the tomato prosystemincDNA. This interpretation is favored by the findings that a homologuecould not be detected at greatly reduced hybridization and washstringencies. Analysis of extracts from other plant genera shouldprovide further insights into the distribution and evolution of membersof the systemin gene family.

EXAMPLE 10 Antisense Suppresion of the Prosystemin Gene.

To determine if the prosystemin gene product has an important role inthe systemic signal transduction leading to the expression of proteinaseinhibitor genes in tomato leaves, a prosystemin antisense DNA wasconstructed and was used to transform tomato plants. The chimericantisense DNA was composed of prosystemin cDNA, in the antisense 3' to5' orientation, under the control of the constitutive CaMV 35S promoterand inserted into the binary vector pGA643.

Materials and Methods:

Strand-specific, radiolabeled RNA probes were produced from theprosystemin cDNA using T3 and T7 RNA polymerases (Stratagene) accordingto the manufacturer's instructions.

The antisense DNA construct was transformed into Agrobacterium strainLBA4404 and the recombinant bacteria were used to transform tomato var.Better Boy. As controls for the primary transformants, tomato plantswere transformed with the binary vector alone. Eighteen antisense plantsand twenty one controls were regenerated. Three weeks after thetransformed plants had been transferred to soil the lower leaves on eachplant were extensively wounded and the levels of wound-inducibleproteinase Inhibitors I and II were determined in the expressed juice ofupper leaves twenty four hours later (C. A. Ryan, 1967, Anal. Biochem.19:434; R. Trautman, K. M. Cown, G. G. Wagner, 1971, Immunochemistry8:901). None of the plants were producing either Inhibitor I orInhibitor II in their leaves prior to wounding. Of the 18 plantscontaining the antisense gene, 11 plants produced Inhibitor I at lessthan 40% of the mean control level of 126.7+/-18.6 μg/ml leaf juice andInhibitor II at less than 30% of the mean control level of 164.7+/-18.6μg/ml leaf juice.

FIG. 16A shows Northern blot analysis of total RNA isolated from one ofthe antisense plants, designated 1A4, using both sense andantisense-specific single-stranded RNA probes. Two samples of total RNA(5 μg) were electrophoresed and blotted as described above. The sampleswere probed separately with radiolabeled RNA probes specific for sense(FIG. 16A, lane 1) and antisense (FIG. 16A, lane 2) prosystemin mRNA(see Examples 6-8, above).

In the wounding experiment the distal leaves of plant 1A4 expressedInhibitor I at 42 μg/ml leaf juice and Inhibitor II at 41 μg/ml leafjuice in response to wounding. The antisense RNA appeared as a band atapproximately 1.7 kilobases (FIG. 16A, lane 2) compared to theprosystemin mRNA at 1 Kb (FIG. 16A, lane 1). southern blot analysisshowed that plant 1A4 contained a single copy of the antisenseconstruct. This conclusion was confirmed by self-fertilizing plant 1A4and analyzing 28F1 progeny by Southern blot analysis. Seven (onequarter) of the 28 F1 progeny did not inherit the antisense construct.This experiment also demonstrated that the antisense construct wasstably inherited in the F1 generation.

To demonstrate that the antisense phenotype segregated with theantisense construct, the levels of Inhibitors I and II in the distalleaves of the 28 F1 plants were measured before wounding, and 24 hrafter wounding. FIG. 16B graphically depicts wound-induced accumulationof proteinase Inhibitor I and FIG. 16C depicts wound-inducedaccumulation of proteinase Inhibitor II in the distal leaves of F1antisense plants (unshaded bars) and untransformed controls (solidbars). Antisense plant 1A4 was self-fertilized and the amounts ofwound-induced proteinase Inhibitors I and II in the distal leaves ofthree-week old F1 progeny were measured by radial immunodiffusion assay(as described below). The plants had an upper and a lower leaf and asmall apical leaf. The lower leaf was wounded and 24 hours later juicewas expressed from the upper, unwounded leaf and assayed. The amount ofInhibitor I was measured in 28 F1 plants while the level of Inhibitor IIwas measured in 27 of the 28 F1 plants. A control group of thirtyuntransformed tomato plants, var. Better Boy, was also wounded and theamounts of Inhibitors I and II were measured. Inhibitor proteins werenot detected in juice expressed from the leaves of six unwoundedantisense plants and six unwounded control plants. Three quarters of theantisense plants (i.e., those inheriting the antisense construct),responded weakly to wounding compared to the control population ofuntransformed plants (FIGS. 16B AND 16C). Plants not inheriting theconstruct produced levels of proteinase inhibitors equal to those of theuntransformed control plants.

In six of the 28 F1 antisense plants Inhibitor I synthesis in the distalleaf was less than 15% of the mean control level of 97.2+/-4.7 μg/mlwhile Inhibitor II synthesis was undetectable in the distal leaf (meancontrol level of 122.3+/-7.2 μg/ml). Southern blot analysis of the sixleast responsive F1 plants suggests that these plants inherited twocopies of the antisense construct, although this conclusion must beconfirmed by self-fertilizing the plants and demonstrating that none ofthe F2 progeny produce proteinase inhibitors in response to wounding atlevels equal to those of the control plants.

These experiments show that expression of antisense prosystemin mRNA intomato inhibits the systemic induction of proteinase inhibitor synthesesin response to wounding. It is inferred that antisense prosystemin mRNAprevents the efficient production of prosystemin and, hence, of themobile systemic wound-signal systemin.

Materials and Methods:

A 747 bp fragment of the prosystemin cDNA was excised from pSYS 1 as aBamHI-Hind III fragment. The BamHI site is located in the bluescriptpolylinker close to the 5'-end of the cDNA, while the Hind III site iswithin the cDNA at nucleotide 859 as numbered in FIGS. 7A and 7B. Theantisense cDNA fragment thus contained all of the prosystemin mRNAsequence except for the first seven bp of the coding region, all of the5'-untranslated region and the last 92 bp of the 3'-untranslated region.The cDNA fragment was placed under the control of the constitutive CaMV35S promoter by cloning it (in the antisense 3' to 5' orientation), intothe polylinker of the binary vector pGA643 digested with Bg1 II and HindIII. The antisense construct was transformed into Agrobacterium strainLBA 4404 and the recombinant bacteria were used to transform tomato var.Better Boy.

Tomato seeds, var. Better Boy, were sterilized by soaking for 15 min ina 15% (v/v) solution of Chlorox containing two or three drops of Tween20. The seeds were washed four times with distilled water then geminatedon medium containing: MS salts (4.3 g/L), agarose (6g/L) and thiamine (1mg/L), pH 5.8. The geminating plants were grown at 28° C. with 16 hrdays. Eighty percent of the seeds germinated. After 7-10 days, when thefirst true leaves appeared, the cotyledons were removed from theseedlings and cut into cubes of edge length 0.2-0.5 cm. The tissue cubeswere preconditioned on tobacco feeder plates for two days at 25° C. inthe dark. Tobacco feeder plates were prepared by subculturing tobacco(NT-1) suspension cells in medium containing: MS sales (4.3 g/L),sucrose (30 g/L), inositol (0.1 g/L), thiamine (1 mg/L), 2, 4-D (0.2mg/L) and KH₂ PO₄ (0.18 g/L) at pH 5.8. The cells were incubated forfour days at 25° C. in the dark. The cells were plated over the samemedium including 0.7% agarose, then incubated under the same conditionsas before for a further two days. Pieces of tomato cotyledon were placedon Whatman No. 4 filter paper soaked in tobacco feeder plate medium andoverlaid onto the tobacco feeder plates. The pieces of preconditionedtissue were punctured with a 20-gauge needle and infected withAgrobacterium by soaking them for thirty min in 15 mL of germinationmedium containing 10⁸ cells/mL. The tissue was blotted dry with sterilefilter paper and incubated on tobacco feeder plates for a further twodays at 25° C. in the dark. The tissue pieces were then washed threetimes in germination medium, the third wash containing 0.5 g/L ofCefotaxime. The tissue pieces were blotted dry with sterile filter paperand placed on shooting medium containing: MS sales (4.3 g/L), thiamine(10 mg/L), nicotinic acid (1 mg/L), pyridoxine (1 mg/L), inositol (100mg/L), sucrose (30 g/L), BAP (2.5 mg/L), IAA (1 mg/L), cefotaxime (250mg/L), carbenicillin (500 mg/L), kanamycin (100 mg/L) and 0.7% (w/v)agarose. The explants were transferred after the first three days ofculture and weekly thereafter. Once callus growth was observed (afterthe third subculture) the explants were transferred to shooting mediumfrom which the IAA and BAP had been removed and zeatin (2 mg/L) added.Once the shoots were 2-3 inches tall they were transferred to rootingmedium which differed from shooting medium in the BAP, cefotaxime andcarbenicillin were absent, vancomycin (0.5 g/L) was added and theconcentrations of sucrose (20 g/L), kanamycin (20 mg/L) and IAA (0.05mg/L) were reduced.

EXAMPLE 11 Effect of Growth on Manduca sexta Larvae Feeding onTransgenic Plants

Northern blot analysis of total RNA extracts, collected at differenttimes from undamaged leaves of control (nontransformed) and transgenictomato plants was performed during feeding experiments with Manducasexta larvae. Plants were approximately 18 inches in height, having 2-3main stems. Total RNA from each sample was separated by electrophoresis,blotted onto nitrocellulose paper and probed with a nick-translated ³²P-prosystemin cDNA. The results are shown in FIG. 17.

The accumulation of Inhibitor I and II proteins in undamaged leaves ofcontrol and trasgenic antisense tomato plants, induced by feeding onManduca sexta larvae was measured. Leaf juice was expressed with amortar and pestle and assayed by radial immunodiffusion assay (Ryan,1967). As shown in FIG. 18, transgenic antisense tomato plants showeddecreased accumulation of both Inhibitor I and II proteins. In FIG. 18,A=transgenic antisense plants, C=wild type plants, PI=Inhibitor I andPII=Inhibitor II.

The growth of Manduca sexta larvae, while feeding on leaves of controland transgenic antisense tomato plants was measured. Ten first instarlarvae were placed randomly on each of six control (wild tape) andtransgenic plants and removed and weighed. Those larvae feeding oncontrol plants were weighed at 14 days and those feeding on transgenicantisense plants were weighed at 10 and 14 days. As shown in FIG. 19,those larvae feeding on transegenic antisense plants showed increasedgrowth weight over those feeding on wild type plants. Values in FIG. 19are the average from three different plant experiments, statisticalsignificance is shown.

Transgenic plants containing a prosystemin gene driven by a CaMVpromoter (Example 10, above) were produced. The prosystemin gene wasessentially the same as the antisense gene (McGurl et al., Science 255,1570-1573), except the prosystemin cDNA was in the sense orientation.Manduca sexta larvae were allowed to feed on leaves of control (wildtype) and transgenic sense tomato plants. The control leaves containedlittle or no Inhibitor I or II. The leaves of the transgenic plantexpressing prosystemin contained about 300 μ/g tissue Inhibitor I andabout 200 μ/g tissue Inhibitor II. As shown in FIG. 20, the weight oflarvae feeding on the control leaves increased at a faster rate than thelarvae feeding on the transgenic sense leaves. Those skilled in the artcan now appreciate from the foregoing description that the broadteachings of the present invention can be implemented in a variety offorms. Therefore, while this invention has been described in connectionwith particular examples thereof, the true scope of the invention shouldnot be so limited since other modifications will become apparent to theskilled practitioner upon a study of the specification and followingclaims.

All publications and applications cited herein are incorporated byreference.

CITATIONS

1. C. A. Ryan, Ann. Rev. Phytopathol. 28, 425 (1990).

2. D. J. Bowles, Ann. Rev. Biochem. 59, 873 (1990).

3. M. Chessin and A. E. Zipf, The Botanical Review 56, 193 (1990).

4. D. L. Dreyer and B. C. Campbell, Plant, Cell and Environ. 10, 353(1987).

5. T. R. Green and C. A. Ryan, Science 175, 776 (1972).

6. C. A. Ryan, TIBS 3, No. 7, 148 (1978).

7. V. A. Hilder, A. M. R. Gatehouse, S. E. Sheerman, R. F. Barker, D.Boulter, Nature 330, 160 (1987).

8. R. Johnson, J. Narvaez, G. An, C. A. Ryan, Proc. Natl. Acad. Sci.U.S.A. 86, 9871 (1989).

9. J. S. Graham, G. Hall, G. Pearce, C. A. Ryan, Planta 169, 399 (1986).

10. J. S. Graham, G. Pearce, J. Merryweather, K. Titani, L. Ericsson, C.A. Ryan, J. Biol. Chem. 260, No. 11, 6555 (1985).

11. J. S. Graham, G. Pearce, J. Merryweather, K. Titani, L. H. Ericsson,C. A. Ryan, J. Biol. Chem. 260, No. 11, 6561 (1985).

12. C. A. Ryan, Plant Physiol. 43, 1880 (1968).

13. W. E. Brown and C. A. Ryan, Biochemistry 23, 3418 (1984).

14. W. E. Brown, K. Takio, K. Titani, C. A. Ryan, Biochemistry 24, 2105(1985).

15. D. Roby, A. Toppan, M. T. Esquerre-Tugaye, Physiol. Mol. Pl. Pathol.30, 6453 (1987).

16. H. D. Bradshaw, J. B. Hoflick, T. J. Parsons, H. R. G. Clarke, PlantMol. Biol. 14,51 (1989).

17. C. A. Ryan and E. E. Fanner, Annu. Rev. Plant. Physiol. Mol.Bio. 42,651 (1991).

18. E. E. Farmer and C. A. Ryan, Proc. Natl. Acad. Sci. U.S.A. 87, 7713(1990).

19. H. Pena-Cortes, J. J. Sanchez-Serrano, R. Mertens, L. Willmitzer, S.Prat, Proc. Natl. Acad. Sci. U.S.A. 86, 9851 (1989).

20. E. Davies, Plant, Cell and Environ. 10, 623 (1987).

21. J. F. Thain, H. M. Doherty, D. J. Bowles, D. C. Wildon, Plant, Celland Environ. 13, 569 (1990).

22. G. Pearce, D. Strydom, S. Johnson, C. A. Ryan, Science 253, 895(1991).

23. B. McGurl, G. Pearce and C. A. Ryan, Plant Molecular Biology,submitted.

24. H. A. Lutcke et al., EMBO Journal 6, 43 (1987).

25. R. B. Harris, Arch. Biochem. Biophys. 275, No. 2, 315 (1989).

26. J. Douglass, O. Civelli and E. Herbert, Ann. Rev. Biochem. 53, 665(1984).

27. L. J. Jung and R. H. Schefler, Science 251, 1330 (1991).

28. C. A. Ryan, Anal. Biochem. 19, 434 (1967).

29. R. Trautman, K. M. Cowan, G. G. Wagner, Immunochemistry 8, 901(1971).

30. T. P. Hopp and K. R. Woods, Proc. Nat. Acad. Sci. 78, 3824 (1981).

31. I. Schechter and A. Berger, Biochem. Biophys. Res. Commun. 27,157(1967).

32. S. O. Rogers and A. J. Bendich, Plant Mol. Biol. 5, 69 (1985).

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 8    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 200 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: polypeptide    (A) DESCRIPTION: prosystemin #1- #200;systemin #179-196;    Figure 6- 7.    (ix) SEQUENCE DESCRIPTION: SEQ ID NO:1:    MetGlyThrProSerTyrAspIleLysAsnLysGlyAspAspMetGln    151015    GluGluProLysValLysLeuHisHisGluLysGlyGlyAspGluLys    202530    GluLysIleIleGluLysGluThrProSerGlnAspIleAsnAsnLys    354045    AspThrIleSerSerTyrValLeuArgAspAspThrGlnGluIlePro    505560    LysMetGluHisGluGluGlyGlyTyrValLysGluLysIleValGlu    65707580    LysGluThrIleSerGlnTyrIleIleLysIleGluGlyAspAspAsp    859095    AlaGlnGluLysLeuLysValGluTyrGluGluGluGluTyrGluLys    100105110    GluLysIleValGluLysGluThrProSerGlnAspIleAsnAsnLys    115120125    GlyAspAspAlaGlnGluLysProLysValGluHisGluGluGlyAsp    130135140    AspLysGluThrProSerGlnAspIleIleLysMetGluGlyGluGly    145150155160    AlaLeuGluIleThrLysValValCysGluLysIleIleValArgGlu    165170175    AspLeuAlaValGlnSerLysProProSerLysArgAspProProLys    180185190    MetGlnThrAspAsnAsnLysLeu    195200    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 951 bases    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (A) DESCRIPTION: prosystemin cDNA; start ATG at #105    (ix) SEQUENCE DESCRIPTION: SEQ ID NO:2:    AAAATTAAATTTGATATTTGGTTTAACTCGATTTTCCATGAACACCCTTAGTGATGAGTA60    TATAAAGCTCAGCTCATGAAGAGTTGAAATAAACTAAGAAAACCATGGGAACTCCTTCAT120    ATGATATCAAAAACAAAGGAGATGACATGCAAGAAGAACCAAAGGTGAAACTTCACCATG180    AGAAGGGAGGAGATGAAAAGGAAAAAATAATTGAAAAAGAGACTCCATCCCAAGATATCA240    ACAACAAAGATACCATCTCTTCATATGTTTTAAGAGATGATACACAAGAAATACCAAAGA300    TGGAACATGAGGAGGGAGGATATGTAAAGGAGAAAATTGTTGAAAAGGAGACTATATCCC360    AATATATCATCAAGATTGAAGGAGATGATGATGCACAAGAAAAACTAAAGGTTGAGTATG420    AGGAGGAAGAATATGAAAAAGAGAAAATAGTTGAAAAAGAGACTCCATCCCAAGATATCA480    ACAACAAAGGAGATGATGCACAAGAAAAACCAAAGGTGGAACATGAGGAAGGAGATGACA540    AAGAGACTCCATCACAAGATATCATCAAGATGGAAGGGGAGGGTGCACTAGAAATAACAA600    AGGTGGTATGTGAGAAAATTATAGTACGAGAAGATCTTGCTGTTCAATCAAAACCTCCAT660    CAAAGCGTGATCCTCCCAAAATGCAAACAGACAATAATAAACTCTAGAAACATCCAAAAA720    AAATTAATAAATAAAAAATTATAATTCAGAACGATAAAGTAAAAATTCTGAATTTGTCTC780    CCGTTAGAAAAGTAACTTCAAATAAATATTTGTCTTTCTTTGTATTTTCAAAGTGTAATT840    TGGTTATTGTACTTTGAGAAGCTTTCTTTAGATTGTTATGTACTTGTATTGCTTCCTTTC900    TTTTGGCTTATTTATATAATATAAATAAAAAATAAATAAATATCTAAAGAT951    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: peptide    (A) DESCRIPTION: systemin; Figure 3.    (ix) SEQUENCE DESCRIPTION: SEQ ID NO:3:    AlaValGlnSerLysProProSerLysArgAspProProLysMetGlnThrAsp    151015    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 4526 bases    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (A) DESCRIPTION: prosystemin genomic DNA; Figures 8A-8C.    (ix) SEQUENCE DESCRIPTION: SEQ ID NO:4:    AAAATTAAATTTGATATTTGGTTTAACTCGATTTTCCATGAACACCCTTAGTGATGAGTA60    TATAAAGCTCAGCTCATGAAGAGTTGAAATAAACTAAGAAAACCATGGGAACTCCTTCAT120    ATGATATCAAAAACAAAGGTATCATTTCTTTATATGCCTAAGTATATATTTATTTATATA180    TTTTGTAACTAAAATTTTATATTAAAATCAACAAGTGAGAGTTTAACAAAAATCATATTA240    AAGAAAAAAAATATATTAATTTTTAATCATGGTATTATCCTCCAGGAGATGACATGCAAG300    AAGAACCAAAGGTGAAACTTCACCATGAGAAGGTAACTTTAGTTTCTCCTTTTTCTTTTT360    TCAACTTCTTTATATATTATTTTTGTAAATTTTTTTATATTATAATGTTCAAATGGTCTC420    ATTTTCTAATTAATAATGTGTCTGAATCGCCATGTTATTTATGTTAGATTTAATACATTA480    ATAACATTGTTAGTAAATGTTAGAATACTGACTCCCAAATTCGCTTAAGGAACAAGTATA540    TTTCATGTGTTTCTTTGCAGATAACAATAATTATGTTTTGTAAAGCAAATAAAATAATAA600    CATAATATTTTATCGTAGAAAACTCCAACTCATTATTTAGATATTTAGATTATGATTTGC660    TTTAATTATACTTTTTTAAACATGATAAATTATTTCTGTTAGACATTTTCGATTCATTTT720    TTTTTTTACAAAAATTGTATTTGCTCTCAAACGTTTACTAGTTAGTTAAGTTAACTATAC780    AAAATATGTCATCTCATTTGATTATATACATCAGGCTCAATTAAAACATATTGGAGATAT840    GGAGATTTTACGATTCATTAACACTAATGTGTATAGTTAGAAAATGTGAAATATTTCAAA900    TGGTTAACTTTTCTGTATAATTGACATTTGAAACTATATGTTTAATTATAACAAACCGTA960    ATCAAATGTTCAAATAAAATTGAATGACAATAGGTATAAGGAGCTATCAATATATTAGCT1020    CTTCTTGATTCAACTTATTTACCGTTATAATTAAATAATGACTCGTTAATTGATTTAATT1080    TTTTTACTCACGTGAAATGATTTAATCAACTCATTTATCACCCTTATTTACGACTCATGT1140    AGAATAATGTTCTTTATACTTGTATACAATTTACTCGGATATTTTTTTTAAATTTTTTTT1200    TATGTTTAATTAAATACTATTAAAATGAAGAAATATTATTTATAATTGAAGAATATTGAA1260    TTTTTTTTCCATCAAAATTTACAGGGAGGAGATGAAAAGGAAAAAATAATTGAAAAAGAG1320    ACTCCATCCCAAGATATCAACAACAAAGATACCATCTCTTCATATGTTTTAAGTATTTAA1380    TTTTTTTCAATCTTTTTTTTTTCTCATCTTCTTATTTTAATCATCTAAAAGAAATTATTA1440    TTATGTTTTTTTTTAACTTTAATTATAATATTATCCAGCAGGAGATGATACACAAGAAAT1500    ACCAAAGATGGAACATGAGGAGGTAACTATATATTTCAATTTATTTACTAATTTATAAAT1560    AATGACTTATTCATTGATTCAATTTATTTTAATTCGTTTGAAATCAAACTAAGGTTACCA1620    TATTATCACCCCACTCCCTCCACTACTCATTTAAAATGATGGTTTGATACTTTGCATGCA1680    ATTTTGTTTATTCATAAGTCATTTATTTTTCAAAAATTTTATGTTCAGTTAAACGTTTGC1740    ATACATTTTGTTTATACATAATTCATCTATTTCTTTTAAAATTTTATGTTCAGTTAAACG1800    ATTGCATACATTTTGTTCATACATAAGTCATCTATTTTTTTAAAAAAAAATTATGTTCAG1860    TTAACGTTTGCATACAATTTTGTTCATACATAATTCATCTATTTTTTTAAAATTTTATGA1920    TCAGTTAAACTTTTATATACAATTTTGCTCGTACATAAGTCATCTATTTTTTTAAAATTT1980    TATGTTCAGTTAAACGTAATAAATAAAATTAGACTGTGGAAATATTATTTATTATTAAAG2040    GATATTACAGGGAGGATATGTAAAGGAGAAAATTGTTGAAAAGGAGACTATATCCCAATA2100    TATCATCAAGATTGAAGGTATAATCTATTTATATGTGTCTAAATATTTAATTTTATTTTT2160    ATTTTTCAGATTTTTTAGTAAGGGATTTTTTTATTTTTTTTCAAAAAATGTGAATCATTT2220    TCAAGAAGTTAATATTATTTTTGGTAACTTTAATCTTGATATATTATTCTCCAGGAGATG2280    ATGATGCACAAGAAAAACTAAAGGTTGAGTATGAGGAGGTAACTTTAATTTCTTCTTTGA2340    CTTTTTATTTATTATTTTTGTATATTTTACTGTCTATTTATTTCATATTCACAAATTATA2400    TTTATCACATATATATTGCTTTATTTTCTTCAAAATTACAGGAAGAATATGAAAAAGAGA2460    AAATAGTTGAAAAAGAGACTCCATCCCAAGATATCAACAACAAAGGTATATATCATATCT2520    TCATATGCCTAAGATTTTATTTTCTTCTTATTTTTCATATTATTTTTATTTAACTAAATT2580    TAGTATGAAACCTTTTTTTTTTTAAAAAAATCATCTTAAATAAAATATTATTTTTGGGTG2640    ACTCAAATCATTGACCTTATATATTCTCCAGGAGATGATGCACAAGAAAAACCAAAGGTG2700    GAACATGAGGTAACTACTTATATTTTTCTCTCTCTTTATTACATAAAATCACATTAGTTA2760    TATGATAATTGGCTATGCTAATAATAAAAAAACAATTAATATATTTATAGGAATTTAAAC2820    AGGGTGGAGTGTCCATGATCTTTATTTTTATCTTGTAAAGTTACTAAGACTATTTCCAAA2880    TAGACCTTTAGTTTGAGCAAAATCTATCAGAAAATACGATAATAAAGAAGTCACGCTGAA2940    AATAAAATATTAATTTTGTGACGTGAAAGCAATATCAAGAGCCCCGTCAATTTGTTGTAT3000    TATGTCAGATGCAACATCCTTCTTTCTTCTCGTGAAGTATAGGAGCGCTTAGCACACATC3060    TCAACATAATGCGCGATAATAACGTTTTAATGGTGAATCTATCGGTATCATAACAATAGT3120    ATACAACTTTAAACCTAATGATCGTCTAGCTAGTAATCTTTCAAAATGAGGGACCCTAAT3180    TACTGACAAAATTTGTGTCTAACATAACTTATGTACCATAACAATAATATATCTTGTGTA3240    ATTTATGAGTGAAGGTAGGGTTTGAAATTAAACATAATCAATAAAATTGGACAAAGAAGA3300    TATTATTTATTAATTGAAAGATATTAATAGTTTTTTTCTTCAAAATTACAGGAAGGAGAT3360    GACAAAGAGACTCCATCACAAGATATCATCAAGATGGAAGGTATCAATCTATTTATATTT3420    TTTATAAGTATTTTTTTCTTACAATTTTTTTATTTCCTTTGGTATATATGAAACTATTTT3480    TTTTAACCATCTTTAAAAAAAAATAATACTTATGTATAACTATAATCATGATATTATCAT3540    CCAGGGGAGGGTGCACTAGAAATAACAAAGGTGGTATGTGAGGTAACTAAATTTCTTCTT3600    CCAATTTTTCTATACATTATGTTTGTATTTTTTTTTTTGGATTCATTCGAACTTTCTTCG3660    ATAGAAAGTCTTGCTATCTATATACGATTAAAATTATATTGAGTTTACGATAAAAATATA3720    TTTAAACAATTCTTTTTTTAATTTCATATCTAAACTATTGAAAATGTGTCTGCCCTCGTA3780    ACCTCGGTACAAAGCCAACTAGAACCACATTTTAAATGATTAAAAAAATCTTTTGAAAGT3840    GTGAGAAATACGCTGAAACTATCGCTTATTATTTTATTTTTACGTATATGCAATAGACAA3900    TATTGAATCCTCTTCTATTTATTCGTATGTTTACTTCCTCACATATCAAATCTCTTAGTA3960    AAAATTCTGACTTCACCACTGTATATATCTTTTTATTTTGATTTTTGATTGCATTTCATT4020    TGTTTAGTTATAATAACTAATAAGGGTCTTTTATTTTATTTATAGCATGATGCTACTATT4080    TTTTGGACACTACAAGGAGCATACAATTCAAATCTCAAACTTTTTTATATTTTTTTTCTA4140    TATTTTTTATTATAAAAGGATATTAATTTCTTTTTTCTTTCAAATACAGAAAATTATAGT4200    ACGAGAAGATCTTGCTGTTCAATCAAAACCTCCATCAAAGCGTGATCCTCCCAAAATGCA4260    AACAGACAATAATAAACTCTAGAAACATCCAAAAAAAATTAATAAATAAAAAATTATAAT4320    TCAGAACGATAAAGTAAAAATTCTGAATTTGTCTCCCGTTAGAAAAGTAACTTCAAATAA4380    ATATTTGTCTTTCTTTGTATTTTCAAAGTGTAATTTGGTTATTGTACTTTGAGAAGCTTT4440    CTTTAGATTGTTATGTACTTGTATTGCTTCCTTTCTTTTGGCTTATTTATATAATATAAA4500    TAAAAAATAAATAAATATCTAAAGAT4526    (2) INFORMATION FOR SEQ ID NO:5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 59 bases    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (A) DESCRIPTION: Figure 11A, con.    (ix) SEQUENCE DESCRIPTION: SEQ ID NO:5:    GAGAATGAAAGAAAAATAGTTGAAAAAGAGACTCCATCCCAAGATATCAACAACAAAGA59    (2) INFORMATION FOR SEQ ID NO:6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 39 bases    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (A) DESCRIPTION: Figure 11B, con.    (ix) SEQUENCE DESCRIPTION: SEQ ID NO:6:    GAGATGATGCACAAGAAAAACCAAAGGTGGAACATGAGA39    (2) INFORMATION FOR SEQ ID NO:7:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 28 bases    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (A) DESCRIPTION: Figure 14, Exon 3.    (ix) SEQUENCE DESCRIPTION: SEQ ID NO:7:    AAGATACCATCTCTTCATATGTTTTAAG28    (2) INFORMATION FOR SEQ ID NO:8:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 27 bases    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA    (A) DESCRIPTION: Figure 14, Exon 7.    (ix) SEQUENCE DESCRIPTION: SEQ ID NO:8:    AAGATATCATATCTTCATATGCCTAAG27    __________________________________________________________________________

What is claimed is:
 1. A polypeptide encoded by a nucleic acidcomprising a nucleotide sequence substantially as shown in SEQ. ID. NO.2.
 2. A polypeptide having an amino acid sequence substantially as shownin SEQ. ID. NO.
 1. 3. A polypeptide having an amino acid sequencesubstantially as shown in SEQ: ID. NO.
 3. 4. A polypeptide comprisingthe amino acid sequence R₁ R₁ QR₁ R₂ PPR₁ R₂ R₂ R₁ PPR₂ R₁ QR₁ R₁,wherein R₁ is any amino acid, R₂ is lysine or arginine, Q is glutamine,and P is proline.
 5. The polypeptide of claim 4, wherein the polypeptidecomprises the amino acid sequence NH₃ -AVQSKPPSKRDPPKMQTD-COO-.
 6. Anamino acid sequence translated from substantially pure systemin mRNA.